Home

EGFr Kit (Clone 31G7) - Thermo Fisher Scientific

1. Endogene Peroxidase Aktivit t wurde unvollst ndig blockiert Unvollst ndige Deparaffinierung berm iger Gebrauch von Gewebefixiermittel Unzureichende Sp lung der Objekttr ger berentwicklung des Substrats Dehydrierung der Probe wahrend der Farbung PRODUKTSPEZIFISCHE GRENZEN 1 Zymeds Maus Anti EGFr Klon 31G7 erfordert eine Enzym Vorbehandlung der FFPE Gewebeschnitte um eine richtige F rbung zu gew hrleisten Auslassung der Vorbehandlung von FFPE Gewebeschnitten kann eine reduzierte F rbung falscher negativer Ergebnisse zur Fogle haben Die Reagenzien wurden optimiert Nicht erneut verd nnen und keine Ersatzreagenzien anderer Herstellerlose verwenden Bei einem Zerfall des Antigens im Gewebe im Verlauf der Zeit sind falsche negative Ergebnisse m glich Die Proben sollten innerhalb von 4 6 Wochen nach der Fixierung auf den Objekttr gern gef rbt werden In diesem Zeitraum m ssen die Proben trocken bei Raumtemperatur 20 25 C aufbewahrt werden Literatur 1 Marquez A et al Evaluation of Epidermal Growth Factor Receptor EGFR by Chromogenic in Situ Hybridization CISH and Immunohistochemistry IHC in Archival Gliomas Using Bright Field Microscopy Diagn Mol Pathol 13 1 1 8 2004 Arita N et al Epidermal Growth Factor Receptor in human glioma J Neurosurg 70 916 919 1989 Quezado MM et al Correlation of EGFR Gene Amplification and Overexpression by Chromogenic In Situ Hybridization CISH and I
2. antig nicit de EGFr Proc dure de coloration tape n 1 Peroxydase Refroidissant Pour des tissus de paraffine incluse ajouter 1 part 30 peroxyde d hydrog ne 9 parts de m thanol absolu Bien m langer Plonger les lames dans une solution de peroxydase refroidissant pendant 10 minutes Laver avec du PBS 2 min 3 fois Passer l tape 2 tape n 2 Digestion enzymatique Pr chauffer la solution de Proteinase K r actif n 1 temp rature ambiante Appliquer 2 gouttes 100 uL de Proteinase K pr chauff e sur chaque sp cimen Incuber pendant 10 min temp rature ambiante Rincer les lames dans du PBS Tween frais pendant 2 min chaque fois tape n 3 Anticorps primaire ou contr le isotype n gatif Eponger l exc s de tampon de la lame Appliquer 2 gouttes 100 uL d anti EGFr de souris r actif n 2 ou de contr le isotype n gatif r actif n 4 sur chaque sp cimen Incuber pendant 30 min Rincer les lames dans du PBS Tween frais pendant 2 min chaque fois tape n 4 de conjugu de polym re IgG anti souris de HRP ponger l exc s de liquide sur la lame Appliquer 2 gouttes 100 uL de conjugu de polym re IgG anti souris de HRP ch vre r actif n 3 sur chaque sp cimen Incuber pendant 10 min Rincer les lames dans du PBS Tween frais pendant 2 min chaque fois Etape n 5 Solution de substrat chromog ne DAB voir la section B3 pour la pr
3. normalmente attesa 3 Controllo tissutale negativo esterno 4 Tessuto di paziente colorato con controllo reagente negativo L assenza di una colorazione marrone specifica nel controllo tissutale negativo conferma la mancata cross reazione del kit con le componenti cellulari Qualora si rilevi una colorazione marrone specifica nel controllo tissutale negativo i risultati ottenuti per il provino del paziente interessato dovrebbero essere ritenuti non validi L assenza di una colorazione marrone specifica indica la specificit della colorazione dell antigene bersaglio da parte dell anticorpo primario Altre eventuali colorazioni marroni rilevate nel provino di un paziente colorato con il controllo reagente negativo a livello di tessuto connettivo leucociti eritrociti o tessuto necrotico dovrebbero essere considerate quali colorazioni di fondo aspecifiche 5 Tessuto di paziente colorato con anticorpo primario Alla rilevazione di una sovraespressione dell EGFr si osserver una colorazione marrone della membrana con o senza colorazione citoplasmatica delle cellule tumorali trattate con anticorpo primario INTERPRETAZIONE DELLA COLORAZIONE Sistema di punteggio dell EGFr di Zymed La proteina EGFr espressa in una gamma variegata di tessuti normali e neoplastici La colorazione delle cellule pu apparire omogenea o eterogenea ovvero colorazione della membrana colorazione citoplasmatica o entr
4. 257 1523 1539 1982 2 Burhow SA et al Affinity labeling of the protein kinase association with the epidermal growth factor receptor in membrane vesicles form A431 cells J Biol Chem 257 40019 40022 1982 3 Chen WS et al Functional independence of the epidermal growth factor receptor from a domain required for ligand induced internalization and calcium regulation Cell 59 33 43 1989 4 Pusztai L et al Growth factors Regulation of normal and neoplastic growth J Pathol 169 191 201 1993 5 Ozane B Richardson CS Overexpression of the EGF is a hallmark of squamous cell carcinoma J Pathol 149 9 14 1986 6 Gusterson B et al Cellular localization of human epidermal growth factor receptor Cell Biol Int Rep 8 649 658 1984 7 Damjanov I et al Immunohistochemical localization of the epidermal growth factor receptor in normal human tissue Lab Invest 55 5 588 592 1986 8 Niikura H et al Expression of epidermal growth factor family proteins and epidermal growth factor receptor in human endometrium Human Pathol 27 3 282 289 1996 9 Rusch V et al Aberrant expression of p53 or the epidermal growth factor receptor is frequent in early bronchial neoplasia and coexpression precedes squamous cell carcinoma development Cancer Res 55 1365 1372 1995 10 Rusch V et al Molecular markers help characterize neuroendocrine lung tumors Ann Thorac Surg 62 798 810 1996 11 Agosti RM et al Expression of the epidermal growth factor recept
5. Die zellul ren Farbungsmuster k nnen homogen oder heterogen ausfallen d h Membranfarbung zytoplasmische Farbung oder Beides Die Verteilung der gef rbten Zellen und die Intensit t der Gewebef rbung kann ebenfalls heterogen sein Zur Bestimmung einer EGFr Protein berexpression kann eine Kombination von Membranf rbungsmuster und Intensit t auf Bais der in Tabelle 3 aufgef hrten Kriterien bewertet werden Zur Unterst tzung der Differenzierung der F rbungsintensit t kann auf die in den Zymed EGFr Bewertungsrichtlinien beiliegenden Farbfotos im Zymed EGFr Kit enthalten Bezug genommen werden 2 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Tabelle 3 EGFr Bewertungssystem f r repr sentative Gewebeschnitte F rbungsmuster Membran F rbungsintensit t Bewertung EGFr Uberexpression Keine F rbung in allen Tumorzellen Keine 0 Negativ Schwache oder kaum wahrnehmbare Sehr Schwach bis 1 Schwach Membranf rbung gef rbt Teilweises Beflecken Schwach im Teil der Membran oder die schwache komplette befleckende Membran wird beobachtet M ige Membranf rbung in der Tumorzellen M ig 2 M ig Starke Membranf rbung in der Tumorzellen Stark 3 Stark Die befleckende EGFr Membran wird normalerweise in den verschiedenen Tumoren wie Kopf und Hals Brust Kolon und Eierstock beobachtet Das cytoplasmatisch Beflecken ist auch in etwas squamous Zelle Krebsen beobachtet worden de
6. 30925 4 6 2005 e Positive und negative Gewebekontrollen unter Qualit tskontrolle auf Seite 4 sind Empfehlungen f r externe Kontrollgewebequellen aufgefiihrt e Farbebecher oder b der e Zeitgeber e Xylen AUFBEWAHRUNG Bei 2 8 C aufbewahren Nicht nach Ablauf des Verfallsdatums verwenden Reagenzien sollten nicht mehr verwendet werden wenn ein Zerfall oder wesentlicher Verlust der Aktivit t zu bemerken sind Im normalen Zustand sind die Reagenzien in diesem Kit eine partikelfreie Fl ssigkeit GEBRAUCHSANLEITUNG A Behandlung der Gewebeschnitte vor der F rbung Zur Erzielung einer optimalen F rbung m ssen die Gewebeschnitte mit einer Proteinase K l sung Reagenz 1 die ebenfalls im Zymed EGFr Kit enthalten ist vorbehandelt werden Bei diesem Verfahren wird die Proteinase K l sung auf Zimmertemperatur erw rmt und dann auf den Gewebeschnitten 10 Minuten bei Zimmertemperatur inkubiert Anweisungen sind unter C3 Schritt 1 des F rbungsverfahrens auf Seite 4 aufgef hrt Bei Verwendung anderer Enzyme kann die Intensit t der EGFr Farbung reduziert oder vollkommen eliminiert werden Durch W rmeeinwirkung induzierte Wiedergewinnung der Epitope HIER darf mit Maus Anti EGFr Klon 31G7 von Zymed nicht eingesetzt werden B Vorbereitung des Reagenz Vor der F rbung m ssen folgende Reagenzien vorbereitet werden B1 Waschpuffer nicht enthalten 10 mL phosphatgepufferte Kochsalzl sung PBS pH 7 4 mit 0 05 Tween 20 B2
7. 6C sont des concentr s de r actifs 20 x Ajouter 1 goutte de r actif 6A 1 goutte de r actif 6B et 1 goutte de r actif 6C 1 mL d eau distill e ou d sionis e Bien m langer Prot ger de la lumi re et utiliser dans l heure Un mL de solution de substrat chromog ne DAB est suffisant pour dix sections de tissus B4 Coloration de contraste non fournie L h matoxyline de Mayer No de cat 00 8001 est recommand e pour la coloration de contraste B5 Solution de fixation non fournie Un support de fixation permanent et non aqueux tel que le Histomount No de cat 00 8030 ou Clearmount No de cat 00 8010 est recommand C Proc dure de coloration Sauf avis contraire tous les r actifs doivent tre quilibr s temp rature ambiante entre 20 et 25 C avant d effectuer l immunocoloration C1 D paraffination et r hydratation 1 Plonger deux fois les lames dans du xyl ne pendant 5 min chaque fois 2 Plonger deux fois les lames dans de l thanol 100 pendant 2 min chaque fois 3 Plonger les lames dans de l thanol 95 pendant 2 min 4 Plonger les lames dans de l thanol 80 pendant 2 min et passer ensuite au refroidissement la peroxydase C2 Protocole d immunohistocoloration Tableau 1 2 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Remarque Ne pas effectuer de restauration antigenique par la chaleur RAC car elle risque d entrainer une perte complete de l
8. Archival Gliomas Using Bright Field Microscopy Diagn Mol Pathol 13 1 1 8 2004 Arita N et al Epidermal Growth Factor Receptor in human glioma J Neurosurg 70 916 919 1989 Quezado MM et al Correlation of EGFR Gene Amplification and Overexpression by Chromogenic In Situ Hybridization CISH and Immunohistochemistry in High Grade Gliomas USCAP Annual Meeting Abstract 1334 2003 Piyathilake CJ et al Differential expression of growth factors in squamous cell carcinoma and precancerous lesions of the lung Clin Cancer Res 8 734 744 2002 Representante autorizado de IVDD 98 79 EC MDSS Burckhardstr 1 D 30163 Hannover Germany Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Z ZYMED Laboratories invitrogen immunodetection 561 Eccles Avenue South San Francisco CA 94080 Tel 800 955 6288 E Mail tech_support invitrogen com Kit EGFr clone 31G7 Syst me de d tection polym re SuperPicTure Pour l immuno coloration du r cepteur du facteur de croissance pidermique Epidermal Growth Factor Receptor EGFr No de cat 28 8763 Bon pour 35 lames tests 1M UTILISATION PREVUE Pour Utilisation de Diagnostiques In Vitro L interpr tation doit tre faite par un pathologiste qualifi dans le cadre des ant c dents cliniques du patient et d autres tests de diagnostics Le kit EGFr de Zymed clone 31G7 est un essai immunohistochimique IHC complet pour l identification de l expression
9. Peroxidase L schl sung nicht enthalten 3 H 0 Zu 1 Teil 3096 Hydrogenperoxid 9 Teile absolutes Methanol hinzugeben Gut vermischen B3 DAB Substrat Chromogenl sung nach dem 3 Protokollschritt auf Seite 4 zubereiten Reagenzien 6A 6B und 6C sind Reagenzien mit 20 facher Konzentration 1 Tropfen Reagenz 6A 1 Tropfen Reagenz 6B und 1 Tropfen Reagenz 6C in 1 mL destilliertes oder entionisiertes Wasser geben Gut vermischen Vor Licht sch tzen und innerhalb einer Stunde verwenden Ein mL DAB Substrat Chromogenl sung reicht f r zehn Gewebeschnitte B4 Gegenf rbung nicht enthalten Mayer H matoxylin Kat Nr 00 8001 wird f r die Gegenf rbung empfohlen B5 Fixativ nicht enthalten Ein wasserfreies permanentes Fixativ wie z B Histomount Kat Nr 00 8030 oder Clearmount Kat Nr 00 8010 wird empfohlen C F rbungsverfahren Wenn nicht anderweitig angegeben sollten alle Reagenzien vor der Immunfarbung an die Raumtemperatur 20 25 C angeglichen werden C1 Entparafinisierung und Rehydrierung 1 Die Objekttr ger zweimal f r jeweils 5 Minuten in Xylen eintauchen 2 Die Objekttr ger zweimal f r jeweils 2 Minuten in 100 Ethanol eintauchen 3 Die Objekttr ger 2 Minuten in 95 Ethanol eintauchen 4 Die Objekttr ger 2 Minuten in 80 Ethanol eintauchen und dann mit der Peroxidase L schung fortfahren 2 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 C2 Immunhistochemisches Farbun
10. Proteinase K Reagente 1 a temperatura ambiente Applicare su ciascun provino 2 gocce 100 uL della Proteinase K preriscaldata Incubare per 10 min a temperatura ambiente Risciacquare in PBS Tween fresca tre volte per 2 min a volta Fase 3 Anticorpo primario o Controllo isotipo negativo Fase 4 Coniugato di polimere igG anti topo HRP Asciugare il liquido in eccesso presente sul vetrino Applicare su ciascun provino 2 gocce 100 uL dell anti EGFr di topo Reagente 2 o del Controllo isotipo negativo Reagente 4 Incubare per 30 min Risciacquare in PBS Tween fresca tre volte per 2 min a volta Asciugare il liquido in eccesso presente sul vetrino Applicare su ciascun provino 2 gocce 100 uL del Coniugato di polimere igG anti topo HRP capra Reagente 3 Incubare per 10 min Risciacquare in PBS Tween fresca tre volte per 2 min a volta Fase 5 Soluzione di substrato cromogeno DAB Per la preparazione del DAB pregasi consultare la sezione B3 Asciugare il liquido in eccesso presente sul vetrino Applicare su ciascun provino 2 gocce 100 uL della Soluzione di substrato cromogeno DAB Reagente 5 Incubare per 5 min Risciacquare con acqua di rubinetto corrente per 3 min Fase 6 Controcolorazione Immergere i vetrini in ematossilina per 1 3 minuti Risciacquare i vetrini con acqua di rubinetto corrente quindi immergerli in acqua ammoniacale diluita allo 0 25 o in PBS a 10 mM pH 7 4 per t
11. absent in the tissue assayed If necessary use a panel of antibodies to identify false negative reactions Cat No 28 8763 PIN 30925 4 6 2005 TRADEMARKS Clearmount HistoGrip Histomount PicTure Peroxo Block and Zymed are trademarks of Zymed Laboratories Inc Authorized Representative MDSS Burckhardstr 1 D 30163 Hannover Germany Authorized Representative for IVDD 98 79 EC 10 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Z ZYMED Laboratories invitrogen immunodetection 561 Eccles Avenue South San Francisco CA 94080 Tel 800 955 6288 E Mail tech_support invitrogen com Equipo EGFr Clon 31G7 Sistema de detecci n de polimerizado SuperPicTure Para la inmunotinci n del receptor del factor de crecimiento epid rmico Epidermal Growth Factor Receptor EGFr Cat N 28 8763 Suficiente para 35 portas pruebas m INDICACIONES Para utilizaci n en diagn stico in vitro La interpretaci n de los resultados debe realizarla un pat logo cualificado dentro del contexto de la historia cl nica del paciente y otras pruebas diagn sticas El equipo Zymed EGFr Clon 31G7 es un an lisis inmunohistoqu mico IHQ completo para la identificaci n de la expresi n de la prote na del EGFr en tejidos normales y neopl sicos que han sido fijados en formalina y infiltrados en parafina para su evaluaci n histol gica Pat logos cualificados deb
12. all materials coming into contact with them should be handled as if capable of transmitting infection and disposed of with proper precautions Never pipette by mouth and avoid contact of antibody and tissue specimens with skin and mucous membranes Minimize microbial contamination of reagents to avoid non specific staining Reagents have been optimally diluted for IHC as described in this package insert Do not further dilute to prevent loss of antigen staining GENERAL LIMITATIONS 1 Immunohistochemistry is a multi step diagnostic process that requires specialized training in the selection of the appropriate reagents tissue selection fixation and processing preparation of the IHC slide and interpretation of the staining results Tissue staining is dependent on the handling and processing of the tissue prior to staining Improper fixation freezing thawing washing drying heating sectioning or contamination with other tissues or fluids may produce artifacts antibody trapping or false negative results Inconsistent results may be due to variations in fixation and embedding methods or to inherent irregularities within the tissue Excessive or incomplete counterstaining may compromise proper interpretation of results The clinical interpretation of any positive or negative staining should be evaluated within the context of clinical presentation morphology and other histopathological criteria The clinical interpretation of positi
13. brune sur la lame de contr le n gatif indique qu une coloration non sp cifique a eu lieu dans l essai Les r sultats de l essai peuvent tre invalides suite une coloration excessive 2 Contr le tissulaire externe positif La pr sence d une coloration brune de la membrane avec ou sans coloration cytoplasmique doit tre observ e comme pr vu pour les contr les tissulaires externes positifs tablis 3 Contr le tissulaire externe n gatif L absence d une coloration brune sp cifique dans le contr le tissulaire n gatif confirme le manque de r activit crois e entre le kit et les composants cellulaires Si une coloration brune sp cifique a lieu dans le contr le tissulaire n gatif les r sultats obtenus pour le sp cimen du patient doivent tre consid r s comme invalides 4 Tissu de patient color avec du contr le de r actif n gatitf Une absence de coloration brune sp cifique indique la sp cificit de la coloration d antig ne cible par l anticorps primaire Toute autre coloration brune ayant lieu dans le sp cimen du patient color avec le contr le de r actif n gatif tel que dans les tissus conjonctifs les leucocytes les rythrocytes ou les tissus n crotiques doivent tre consid r s comme tant une coloration de fond non sp cifique 5 Tissu de patient color avec des anticorps primaires Lorsqu une surexpression d EGFr est d tect e elle ressembler
14. citoplasm tica en gliomas y las carcinomas de p lmon 4 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 CARACTERISTICAS DE RENDIMIENTO Especificidad La expresi n de EGF4 en c lulas A431 carcinoma epidermoide humano ya sea estimulado o no estimulado con EGF ha sido detectada por el anticuerpo anti EGFr de rat n clon 31G7 En an lisis por inmunotransferencia el clon 31G7 reconoci a la banda de prote na kDa 170 correspondiente al peso molecular publicado para el EGFr Clon 31G7 no reacciona con HER2 Clon 31G7 reconocer EGFr vIII Reproducibilidad El equipo Zymed EGFr se someti a pruebas en 57 casos de muestras tisulares de carcinoma de cabeza y cuello Cada caso examinado demostr un acuerdo regular de la reproducibilidad en la evaluaci n de la tinci n en comparaci n con la falta de ella Inmunoreactividad Reactividad del tejido anormal Todos los estudios mencionados a continuaci n utilizaron el anticuerpo del clon 31G7 y tejidos infiltrados en parafina y fijados en formalina La sobre expresi n del EGFr fue demostrada en neoplasias pulmonares En un estudio de 92 tumores neuroendocrinos el 28 de las muestras de carcinoide t pico el 57 de las de carcinoide at pico el 42 de las de carcinoma neuroendocrino de c lulas grandes el 3996 de las de carcinoma neuroendocrino mixto de las c lulas pequefias y grandes y el 22 de las muestras de carcinoma pulmonar de las c lulas pequefias demost
15. false negative results Reagents have been optimized Do not further dilute or substitute reagents of different manufactured lots False negative results may be obtained due to the degradation of the antigen in tissues over time Specimens should be stained within 4 6 weeks after mounting on slides when stored at dry room temperature 20 25 C SAFETY AND PRECAUTIONS l 2 3 For In Vitro Diagnostic Use Use ample precautions when handling reagents Wear disposable gloves coat and safety glasses when handling suspected carcinogens Do not use this kit after expiration date After use store as specified Any storage conditions other than those specified by this package insert must be validated by the user Avoid contact of eyes and mucous membranes with reagents If reagents come into contact with sensitive areas flush with generous amounts of water 3 3 Diaminobenzidine tetrachloride DAB may be harmful if swallowed inhaled or absorbed through the skin and may be irritating to eyes skin mucous membranes and upper respiratory tract DAB is a suspected carcinogen consult Federal State and or local regulations for disposal recommendations The 0 1 sodium azide NaN used as a preservative in this kit is toxic if ingested Sodium azide may react with lead and copper plumbing to form highly explosive metal azides To prevent azide build up in plumbing flush with large volumes of water during disposal Patient specimens and
16. hin Die Assay Ergebnisse sind eventuell aufgrund einer berm igen F rbung ung ltig 2 Externe positive Eine braune Membranfarbung mit oder ohne zytoplasmischer F rbung Gewebekontrolle sollte gem der Erwartungen f r etablierte externe positive Gewebekontrollen beobachtet werden 3 Externe negative Die Abwesenheit einer braunen spezifischen Farbung in der negativen Gewebekontrolle Gewebekontrolle best tigt die fehlende Kreuzreaktion des Kits auf zellul re Komponenten Wenn die negative Gewebekontrolle eine braune spezifische F rbung aufweist sollten die f r die Patientenprobe gewonnen Ergebnisse als ung ltig betrachtet werden 4 Mit negativer Die Abwesenheit der braunen spezifischen Farbung zeigt die Spezifitat Reagenzkontrolle gef rbtes der Zielantigenf rbung durch den prim ren Antik rper an Patientengewebe Andere braunen Farbungen in der mit negativer Reagenzkontrolle gef rbten Patientenprobe z B in Bindegewebe Leukozyten Erythrozyten oder nekrotischem Gewebe sollten als nicht spezifische Hintergrundfarbung betrachtet werden 5 Mit prim rem Antik rper Eine EGFr berexpression zeigt sich als braune Membranf rbung mit gef rbtes Patientengewebe oder ohne zytoplasmischer F rbung der Tumorzellen die mit prim rem Antik rper behandelt wurden INTERPRETATION DER FARBUNG Zymed EGFr Bewertungssystem EGFr Protein wird in verschiedenen normalen und neoplastischen Geweben ausgedr ckt
17. porta contiene 2 cortes de estirpes celulares infiltrados en parafina y fijados en formalina que representan la expresi n de niveles positivos 24 y negativos de la prote na del EGFr Recomendaciones para la evaluaci n de EGFr un juego de fotos Se incluyen dos juegos de fotograf as color uno con muestras de tejido carcinoma de colon como recomendaciones para la clasificaci n REACTIVOS Y MATERIALES NECESARIOS PERO NO PROPORCIONADOS e Metanol absoluto e Cubreobjetos e Agua destilada o desionizada e Etanol e Hematoxilina de Mayer e Per xido de hidr geno al 30 e Microscopio de luz e Medio de montaje como Clearmount Cat N 00 8010 o Histomount Cat N 00 8030 e 10mm de soluci n salina amortiguada fosfato PBS pH 7 4 con Tween 20 al 0 05 1 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 e Controles tisulares positivos y negativos consulte el control de calidad p gina 4 para leer las recomendaciones especificas sobre las fuentes externas de control tisular e Jarras o ba os de tinci n e Cron metro e Xileno ALMACENAMIENTO Almacenar a 2 8 C No utilizar despu s de la fecha de caducidad No utilizar los reagentes si el deterioro o la p rdida sustancial de actividad es evidente La apariencia normal de los reactivos que forman parte de este equipo es la de un l quido libre de part culas INSTRUCCIONES DE USO A Pretratamiento de los cortes tisulares antes de l
18. uso della Ematossilina di Mayer N Cat 00 8001 B5 Soluzione di fissaggio non acclusa Si raccomanda l impiego di un mezzo di fissaggio permanente non acquoso quale Histomount N Cat 00 8030 o Clearmount N Cat 00 8010 C Procedura di colorazione Salvo indicazione contraria tutti i reagenti dovrebbero essere equilibrati a temperatura ambiente 20 25 C prima dell immunocolorazione C1 Deparaffinizzazione e reidratazione 1 Immergere i vetrini in xilene due volte per 5 min alla volta 2 Immergere i vetrini in etanolo al 100 due volte per 2 min alla volta 3 Immergere i vetrini in etanolo al 95 per 2 min 4 Immergere i vetrini in etanolo all 80 per 2 min quindi eseguire il quenching di perossidasi 2 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 C2 Protocollo di immunoistocolorazione Tabella 1 Nota Non eseguire il richiamo dei determinanti antigeni indotto mediante calore Heat Induced Epitope Retrieval HIER giacch ci potrebbe causare la perdita completa dell antigenicit EGFr Procedura di colorazione Fase 1 Quenching di perossidasi Per i tessuti inclusi in paraffina aggiungere il 30 di perossido di idrogeno a 9 parti di metanolo assoluto Mescolare bene Immergere vetrini in soluzione quenching di perossidasi per 10 minuti Lavare con PBS 2 min 3 volte Procedere al Passaggio 2 Fase 2 Digestione enzimatica Preriscaldare la soluzione a base di
19. 7 the sequential application of an HRP Goat anti Mouse IgG polymer conjugate and then DAB chromogen allows the localization of the bound primary antibody within tissues Peroxidase catalyzes the substrate hydrogen peroxidase converting the chromogen DAB into a permanent brown deposit The specimen may then be counterstained and coverslipped and staining results visualized under a light microscope Control slides containing formalin fixed paraffin embedded cell lines demonstrating positive and negative EGFr expression are provided as part of this kit to validate staining performance in each IHC assay REAGENTS PROVIDED Good for 35 Tests Reagent 1 One dropper bottle 8 mL of Ready To Use Proteinase K solution Reagent 2 One dropper bottle 4 mL of Ready To Use Mouse anti EGFr Clone 31G7 antibody Reagent 3 One dropper bottle 4 mL of Ready To Use HRP Goat anti Mouse IgG polymer conjugate Reagent 4 One dropper bottle 4 mL of Ready To Use Mouse IgG Negative Isotype Control Reagent 5A One dropper bottle 1 mL of 20X Buffer concentrate Reagent 5B One dropper bottle 1 mL of 20X DAB chromogen Reagent 5C One dropper bottle 1 mL of 20X 0 6 Hydrogen peroxide Control Slides 5 Control Slides unstained Each slide contains 2 formalin fixed paraffin embedded cell line sections representing positive 2 and negative levels of EGFr protein expression EGFr Evaluation Guidelines 1 photo set One set of color photographs i
20. Abnormal Tissue Reactivity All studies cited below used clone 31G7 antibody and formalin fixed paraffin embedded tissues EGFr over expression has been demonstrated in pulmonary neoplasms In one study of 92 neuroendocrine tumors 28 of typical carcinoid 57 of atypical carcinoid 42 of large cell neuroendocrine carcinoma 39 of mixed small and large cell neuroendocrine carcinoma and 22 of small cell lung carcinoma demonstrated a score of 2 to 4 in EGFr staining In another study EGFr over expression was observed in 56 of lung adenocarcinoma and 84 of lung squamous cell carcinoma specimens Clone 31G7 stained 50 of head and neck squamous cell carcinoma specimens in one study Variable EGFr expression in breast carcinoma specimens has been reported despite its presence on cells derived from all three germ layers one study reported EGFr overexpression in up to 35 of breast cancers Clone 31G7 has also been used in studies of glandular cells of endometrial and prostatic carcinoma In one study of 40 cases of endometrioid endometrial carcinoma Mouse anti EGFr clone 31G7 exhibited positive staining in 65 of the specimens 6 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Normal Tissue Reactivity Table 4 reflects normal tissue reactivity exhibited by Mouse anti EGFr clone 31G7 Tissues were formalin fixed paraffin embedded and stained with clone 31G7 according to the instructions in the EGFr Kit Tab
21. Contra tinci n Sumergir los portas en hematoxilina durante 1 a 3 minutos Enjuagar los portas en agua corriente luego sumergirlos en amonfaco al 0 25 diluido en agua o 10 mm PBS pH 7 4 para colorear los n cleos de azul Nota la contra tinci n producir la coloraci n azul claro a azul oscuro de los n cleos celulares dependiendo del tiempo de incubaci n y de la potencia de la hematoxilina La contra tinci n excesiva o incompleta puede comprometer la correcta interpretaci n de los resultados Paso 7 Cubreobjetos Enjuagar brevemente los portas en agua destilada o desionizada Deshidratar los portas en una serie graduada de etanol al 70 80 95 y 100 luego eliminar una vez en xileno Se recomienda la utilizaci n de un medio de montaje permanente y no acuoso Aplicar al porta 2 gotas 100 uL de soluci n de montaje Histomount o Clearmount luego agregar el cubreobjetos CONTROL DE CALIDAD Portaobjetos de control El equipo Zymed EGFr contiene 5 portaobjetos de control sin tinci n Cada portaobjetos contiene 2 cortes de estirpes celulares infiltrados en parafina y fijados en formalina que representan la expresi n de niveles positivos 2 y negativos de la prote na del EGFr A fin de demostrar la validez de la ejecuci n de la tinci n se debe colorear un portaobjetos de control en cada procedimiento de tinci n EGFr Porta de Control Imagen 1 Porta de control EGFr con positivo 24 est
22. Expressionen anzeigen sind f r die Validierung der F rbleistung jedes IHC Assay im Kit enthalten MITGELIEFERTE REAGENZIEN Ausreichend fiir 35 Tests Reagenz 1 Ein Tropferfl schchen 8 mL gebrauchsfertige Proteinase K l sung Reagenz 2 Ein Tropferflaschchen 4 mL gebrauchsfertiger Anti EGFr Klon 31G7 Mausantik rper Reagenz 3 Ein Tropferflaschchen 4 mL gebrauchsfertiges HRP Ziegen Anti Maus IgG Polymerkonjugat Reagenz 4 Ein Tropferflaschchen 4 mL gebrauchsfertige Maus IgG negative Isotypkontrolle Reagenz 5A Ein Tropferflaschchen 1 mL Pufferkonzentrat 20X Reagenz 5B Ein Tropferfl schchen 1 mL DAB Chromogen 20X Reagenz 5C Ein Tropferflaschchen 1 mL 0 6 Hydrogenperoxid 20X Kontrollobjekttr ger 5 Kontrollobjekttr ger ungef rbt Jeder Objekttr ger enth lt 2 in Formalin fixierte in Paraffin eingebettete Zellreihenschnitte f r positive 2 und negative Levels der EGFr Proteinexpression EGFr Bewertungsrichtlinien 1 Foto Als Richtlinien fiir die Bewertung eines Kolonkarzinoms ERFORDERLICHE JEDOCH NICHT MITGELIEFERTE REAGENZIEN UND MATERIALIEN Absolutes Methanol Coverslips Destilliertes oder deionisiertes Wasser Ethanol Mayer H matoxylin 30 Hydrogenperoxid Lichtmikroskop Fixativ wie z B Clearmount Kat Nr 00 8010 oder Histomount Kat Nr 00 8030 10 mL phosphatgepufferte Kochsalzl sung PBS pH 7 4 mit 0 05 Tween 20 1 Zymed Laboratories Inc Cat No 28 8763 PIN
23. Reagent Control Absence of brown specific staining indicates the specificity of target antigen staining by the primary antibody Other brown staining occurring in the patient specimen stained with the negative reagent control as in connective tissue leucocytes erythrocytes or necrotic tissue should be considered non specific background staining 5 Patient Tissue Stained With When EGFr over expression is detected it will appear as brown Primary Antibody membrane staining with or without cytoplasmic staining of tumor cells that have been treated with primary antibody 5 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 INTERPRETATION OF STAINING Zymed EGFr Grading System The EGFr protein is expressed in a variety of normal and neoplastic tissues Cellular staining pattern may be homogenous or heterogeneous ie membrane staining cytoplasmic staining or both The distribution of stained cells and the intensity of tissue staining may also be heterogeneous For the determination of EGFr protein over expression a combination of membrane staining pattern and intensity can be evaluated based on criteria in Table 3 To aid in the differentiation of staining intensity please refer to the color photographs in Zymed EGFr Evaluation Guidelines provided with Zymed EGFr Kit Table 3 EGFr Scoring System for Representative Tissue Section Staining Pattern Membrane Staining Intensity Score EGFr Ove
24. Z ZYMED Laboratories invitrogen immunodetection 561 Eccles Avenue South San Francisco CA 94080 Tel 800 955 6288 E Mail tech_support invitrogen com EGFr Kit Clone 31G7 SuperPicTure Polymer Detection For Immunohistostaining of Epidermal Growth Factor Receptor EGFr Cat No 28 8763 Good for 35 Slides Tests INTENDED USE For In Vitro Diagnostic Use Zymed EGFr Kit Clone 31G7 is an immunohistochemical IHC assay for the identification of EGFr protein expression in normal and neoplastic tissues that have been formalin fixed and paraffin embedded for histological evaluation Interpretation of test results must be made within the context of the patient s clinical history and in conjunction with other diagnostic tests by qualified pathologists SUMMARY AND EXPLANATION Background The Epidermal Growth Factor Receptor EGFr HER1 c erbB 1 is a 170 kDa membrane protein that consists of an extracellular EGF binding domain a short transmembrane region and an intracellular domain with ligand activated tyrosine kinase activity EGFr has two common ligands epidermal growth factor EGF and Transforming Growth Factor alpha TGF a Binding of TGF a activates the EGFr signaling cascade with EGFr mediated phosphorylation of substrates leading to an increase in cytosolic calcium ions in target cells increased DNA synthesis and proliferation and differentiation of the cell TGF a is an important growth factor in the tra
25. a une coloration brune de la membrane avec ou sans coloration cytoplasmique des cellules tumorales trait es avec un anticorps primaire INTERPRETATION DE LA COLORATION Syst me de notation de EGFr de Zymed La prot ine EGFr est exprim e dans une gamme de tissus normaux et n oplasiques Le motif de la coloration cellulaire peut tre homog ne ou h t rog ne c d une coloration de la membrane une coloration cytoplasmique ou les deux La distribution de cellules color es et l intensit de la coloration du tissu peuvent galement tre h t rog nes Pour d terminer la surexpression de la prot ine EGFr une combinaison du motif et de l intensit de la coloration de la membrane peut tre valu e en se basant sur les crit res du Tableau 3 Pour aider diff rencier les intensit s de coloration veuillez vous reporter aux photos en couleurs dans les Directives d valuation de l EGFr de Zymed fournies avec le kit EGFr de Zymed Tableau 3 Syst me de notation de EGFTr pour une section de tissu repr sentative Motif de coloration Membrane Intensit de coloration Note Surexpression de EGFr Pas de coloration dans toutes les cellules tumorales Aucun 0 N gatif Une coloration faible ou peine visible de la Tr s Faible Faible 1 Faible membrane est observ e Une partie de la membrane est color e ou la coloration de la membrane complete est faible Une coloration mod
26. a tinci n Para una tinci n ptima los cortes tisulares se deben pretratar con soluci n de proteinase K reactivo 1 que se incluye en el equipo Zymed EGFr Este procedimiento incluye el precalentamiento de la soluci n de proteinase K a temperatura ambiente seguido de la incubaci n de la soluci n de proteinase K a temperatura ambiente durante 10 minutos en los cortes tisulares para instrucciones consulte C3 paso 1 del procedimiento de tinci n p gina 4 El tratamiento con otras enzimas puede reducir o eliminar por completo la intensidad de la tinci n de EGFr La recuperaci n del ep topo inducida por el calor HTER no puede ser utilizada con el anti EGFr de rat n clon 31G7 de Zymed B Preparaci n del reactivo Antes de proceder a la tinci n preparar los siguientes reactivos B1 Amortiguador para lavado no se proporciona con el equipo 10 mm de soluci n salina amortiguada fosfato PBS pH 7 4 con Tween 20 al 0 05 B2 Soluci n para extinci n de la peroxidasa no se proporciona con el equipo H O al 3 Agregar parte de per xido de hidr geno al 30 a 9 partes de metanol absoluto Mezclar bien B3 Soluci n sustrato crom geno DAB preparar despu s del paso 3 del protocolo en la p gina 4 Los reactivos 6A 6B y 6C son reactivos concentrados 20X Agregar gota de reactivo 6A de reactivo 6B y 1 de reactivo 6C a 1 mL de agua destilada o desionizada Mezclar bien Proteger de la luz y utilizar en el plazo de una ho
27. ains 5 control slides unstained Each control slide contains 2 formalin fixed paraffin embedded cell line sections representing positive 2 and negative levels of EGFr protein expression One control slide should be stained along with each staining procedure to demonstrate the validity of the staining run EGFr Control Slide Image 1 EGFr Control Slide with positive 2 cell line A and negative cell line B Please note that cell lines are not drawn to scale and are smaller than indicated in the diagram Positive Tissue Control External positive control materials should be taken from fresh autopsy biopsy surgical specimens that have been fixed processed and embedded in the same manner as the samples Positive tissue controls are indicative of correctly prepared tissues and proper staining techniques One positive tissue control for each set of test conditions should be included in each staining run Specimens processed differently from the patient samples validate reagent performance only and do not verify tissue preparation 4 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Colorectal carcinoma may be useful as a source of positive control tissue Known positive tissue controls should only be utilized for monitoring the correct performance of processed tissues and test reagents rather than as an aid in formulation of a specific diagnosis for patient samples If the positive tissue controls fail to demo
28. aires fix es dans du formol et incorpor es dans de la paraffine repr sentant des niveaux d expression de prot ine EGFr positifs 2 et n gatifs Une lame de contr le doit tre color e pour chaque proc dure de coloration afin d illustrer la validit de l essai de coloration Lame de contr le EGF Image 1 Lame de contr le EGFr avec lign e cellulaire A positif 2 et lign e cellulaire B n gatif Veuillez noter cela lign es cellulaires ne sont pas dessin s la balance et soyez plus petit qu indiqu dan le diagremme Les cancers colorectaux chez l homme peuvent tre utiles comme sources de tissus de contr le positifs Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Toute coloration douteuse doit tre signal e au Service technique de Zymed Laboratories Inc Le kit EGFr de Zymed n a pas t con u pour fournir des informations de diagnostic au patient et ou au m decin et n a pas t valid a ces fins Les lames doivent tre valu es en suivant le Tableau 2 afin de v rifier la validit de chaque s rie de coloration Tableau 2 Evaluation des lames de contr le et des lames d chantillons Ordre de lecture des lames Evaluation 1 Lames de contr le fournies Pour v rifier la validit du souillure couru La pr sence d une coloration de la membrane brune positive sur la lame de contr le positif indique un essai valide La pr sence d une coloration
29. ambe Anche la distribuzione delle cellule colorate e l intensit della colorazione tissutale potrebbero apparire eterogenee Per la determinazione della sovraespressione della proteina EGFr si pu eseguire una valutazione combinata del modello e dell intensit della colorazione attenendosi ai criteri riportati nella Tabella 3 Per agevolare la differenziazione dell intensit della colorazione pregasi far riferimento alle fotografie a colori riportate nelle Linee guida Zymed per la valutazione dell EGFr accluse al Kit EGFr Zymed Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Tabella 3 Sistema di punteggio dell EGFr per sezioni tissutali rappresentative Modello di colorazione Membrana Intensita di Punteggio Sovraespressione colorazione dell EGFr Nessuna colorazione in tutte le cellule tumorali Nessuna 0 Negativo Si osserva una colorazione lieve o appena percettibile Molto Debole 1 Debole della membrana Colorazione solo parziale della a Debole membrana oppure una debole colorazione totale della membrana Si osserva una colorazione moderata della membrana Moderata 2 Moderata delle cellule tumorali Si osserva una forte colorazione della membrana delle Forte 3 Forte cellule tumorali Solitamente in vari tumori quali mammella colon ovario testa e collo si osserva una colorazione di membrana per l EGFr Nel glioma si sono talvolta evidenziate marcatur
30. ambos La distribuci n de las c lulas coloreadas y la intensidad de la tinci n tisular tambi n pueden ser heterog neas Para la determinaci n de la sobre expresi n de la prote na del EGFr solamente la membrana que se mancha debe ser considerada Consulte las fotograf as color en las recomendaciones para la evaluaci n de EGFr de Zymed proporcionado con el equipo Zymed EGFr Tabla 3 Sistema de puntuaci n del EGFr para cortes tisulares representativos Patr n de tinci n Membrana Intensidad de Resultado Sobre expresi n tinci n de EGFr No hay tinci n en las c lulas tumorales Nada 0 Negativo Se observa una tinci n de membrana d bil o apenas D bil a Muy 1 D bil perceptible en las c lulas tumorales Se observa una D bil tinci n en parte de la membrana o tinci n d bil de toda la membrana Se observa la tinci n moderada de la membrana en Moderada 2 Moderada las c lulas tumorales Se observa una tinci n fuerte de la membrana en las Fuerte 3 Fuerte c lulas tumorales La tinci n EGFr de la membrana es observado usualmente en varios tumors como cabeza y cuello pecho colon y ovario Sin embargo la tinci n membrana y citoplasm tica ha serado observado o solo o en combinaci n an glioma La tinci n citoplasm tica ha serado observado en algunas carcinomas de las c lulas escamosas del pulm n Por lo tanto puede ser importante a informar la intensidad de la tinci n
31. boratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Z ZYMED Laboratories invitrogen immunodetection 561 Eccles Avenue South San Francisco CA 94080 Tel 800 955 6288 E Mail tech_support invitrogen com Kit EGFr Clone 31G7 Sistema di rilevazione polimere SuperPicTure Per l immunocolorazione del recettore per il fattore di crescita dell epidermide Epidermal Growth Factor Receptor EGFr N Cat 28 8763 Sufficiente per 35 vetrini test d SCOPO D UTILIZZO Per uso diagnostico In Vitro L interpretazione deve essere effettuata da un patologo qualificato entro il contesto dell anamnesi clinica del paziente ed in considerazione di altri test diagnostici Il Kit EGFr di Zymed Clone 31G7 costituisce una prova immunoistochimica IIC per l identificazione dell espressione della proteina EGFr nei tessuti normali e neoplastici fissati in formalina ed inclusi in paraffina ai fini della valutazione istologica L interpretazione degli esiti dell analisi deve essere effettuata da un patologo qualificato entro il contesto dell anamnesi clinica del paziente ed in considerazione di altri test diagnostici Principio di funzionamento Il Kit EGFr di Zymed prevede l impiego dell anticorpo anti EGFr di topo clone 31G7 e della metodologia di rilevazione SuperPicTure polimere brevettata da Zymed per rilevare la presenza di anticorpi primari legati ad antigeni in provini fissati in formalina ed inclusi in paraffina Al presente
32. colo de quanto indicto nello schema Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 I campioni di tessuto affetto da carcinoma colon rettale potrebbero risultare utili quali fonti per il controllo tissutale positivo Qualsiasi colorazione controversa dovrebbe essere comunicata al Reparto di assistenza tecnica Zymed Laboratories Inc Lo scopo del Kit EGFr di Zymed non consiste nel fornire a pazienti e o medici informazioni prognostiche e non ne stato convalidato l uso per tale scopo I vetrini dovrebbero essere valutati conformemente a quanto riportato nella Tabella 2 ai fini della conferma della validita di ciascuna procedura di colorazione Tabella 2 Valutazione del vetrino di controllo e del vetrino campione Ordine di lettura dei vetrini 1 Vetrini di controllo Acclusi Per verificare la validita della macchiatura serie Valutazione La presenza di una colorazione marrone positiva della membrana nel vetrino del controllo positivo indica una prova valida La presenza di una colorazione marrone nel vetrino del controllo negativo indica che nella prova in oggetto si verificata una colorazione aspecifica I risultati di una prova potrebbero risultare non validi a causa di una colorazione eccessiva 2 Controllo tissutale positivo esterno Per i controlli tissutali positivi esterni standard si dovrebbe poter osservare la colorazione marrone della membrana con o senza colorazione citoplasmatica
33. de la prot ine EGFr dans des tissus normaux et n oplasiques fix s dans du formol et incorpor s dans de la paraffine pour une valuation histologique L interpr tation des r sultats examens doit tre effectu e dans le cadre des ant c dents cliniques du patient et simultan ment avec d autres tests de diagnostic effectu s par des pathologistes qualifi s Principe de proc dure Le kit EGFr de Zymed utilise l anticorps de souris anti EGFr clone 31G7 et la m thodologie de d tection SuperPicTure brevet e de Zymed pour d tecter la pr sence d anticorps primaires attach s aux antig nes dans des sp cimens fix s dans du formol et incorpor s dans de la paraffine Des lames de contr le contenant des lign es cellulaires fix es dans du formol et incorpor es dans de la paraffine manifestant une expression d EGFr positive et n gative font partie de ce kit afin de valider la performance de coloration dans chaque essai IHC REACTIFS FOURNIS Bons pour 35 tests R actif n 1 Un flacon compte gouttes 8 mL de solution de Proteinase K pr te l emploi R actif n 2 Un flacon compte gouttes 4 mL d anticorps anti EGFr clone 31G7 de souris pr t l emploi R actif n 3 Un flacon compte gouttes 4 mL de conjugu de polym re IgG anti souris de HRP ch vre R actif n 4 Un flacon compte gouttes 4 mL de contr le isotype n gatif d IgG de souris pr t l emploi R actif n 5A Un flacon compte gouttes 1 mL de concent
34. di carcinoma neuroendocrino a grandi cellule il 39 dei casi di carcinoma neuroendocrino a cellule miste piccole e grandi e il 22 dei casi di carcinoma polmonare a piccole cellule hanno riportato un punteggio di colorazione EGFr compreso tra 2 e 4 In un altro studio la sovraespressione dell EGFr stata osservata nel 56 dei campioni tratti da soggetti affetti da adenocarcinoma polmonare e nell 84 dei campioni derivati da pazienti affetti da carcinoma polmonare a cellule squamose In uno studio il clone 31G7 ha colorato il 50 dei campioni di tessuto affetto da carcinoma a cellule squamose della testa e del collo In alcuni campioni di tessuto affetto da carcinoma mammario si rilevata una espressione dell EGFr variabile nonostante ne sia stata osservata la presenza in cellule derivate da tutti e tre gli strati germinativi in uno studio la sovraespressione dell EGFr stata rilevata fino al 35 dei casi di carcinoma mammario Il clone 31G7 inoltre stato usato in studi condotti su cellule ghiandolari affette da carcinoma endometriale e prostatico In uno studio condotto su 40 casi di carcinoma endometriale endometrioide l anti EGFr di topo clone 31G7 ha manifestato una colorazione positiva nel 65 dei campioni RISOLUZIONE DEI PROBLEMI Cause possibili di colorazione negativa nei vetrini positivi 1 Le fasi previste dal protocollo di colorazione non sono state eseguite nell ordine corretto 2 Sono state omesse le fasi
35. di incubazione dell anticorpo primario e secondario 3 Sono stati distrutti degli anticorpi labili 4 I provini non sono stati debitamente fissati e o trattati 5 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 5 I provini si sono disidratati nel corso della colorazione Cause possibili di colorazione debole in tutti i vetrini 1 2 3 4 I provini hanno trattenuto il liquido in eccesso accumolatosi durante le fasi di risciacquo Sono stati osservati tempi di incubazione insufficienti Il substrato non stato preparato correttamente Deparaffinizzazione incompleta la colorazione potrebbe essere accompagnata da fondo elevato Cause possibili di colorazione con fondo elevato Dw Pah te L attivit perossidasica endogena non stata bloccata completamente Deparaffinizzazione incompleta Applicazione di un quantitativo eccessivo di adesivo per tessuti Risciacquo inadeguato dei vetrini Sovrasviluppo del substrato Disidratazione dei provini durante la colorazione LIMITAZIONI SPECIFICHE DEL PRODOTTO 1 Ai fini della debita colorazione l anti EGFr di topo clone 31G7 di Zymed richiede il pretrattamento enzimatico delle sezioni tissutali fissate in formalina ed incluse in paraffina formalin fixed paraffin embedded FFPE Il mancato pretrattamento delle sezioni tissutali FFPE potrebbe comportare una riduzione della colorazione dei risultati falsi negativi I reagenti sono stati ottimizzat
36. e 2 5 Tonsil 5 Squamous epithelial cells 3 3 5 Negative 2 5 Staining pattern Both membrane and cytoplasmic staining may be observed t Myoepithelial cells muscularis mucosa in different tissues may be stained 1 to 2 in some cases TROUBLESHOOTING Possible causes for negative staining on positive slides SA a ai Steps in the staining protocol were performed in incorrect sequence Primary or secondary antibody incubation steps were omitted Labile antigens were destroyed Specimen was improperly fixed and or processed Specimen dehydrated during staining Possible causes for weak staining on all slides 1 Specimen retained excess liquid after rinsing steps 2 Incubation times were insufficient 3 Substrate prepared improperly 4 Deparaffinization was incomplete staining may be accompanied by high background Possible causes for high background staining Endogenous peroxidase activity was incompletely blocked 2 Deparaffinization was incomplete 3 Excessive application of tissue adhesive Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 4 5 6 Inadequate rinsing of slides Over development of substrate Dehydration of specimen during staining PRODUCT SPECIFIC LIMITATIONS 1 2 3 Zymed Mouse anti EGFr clone 31G7 requires enzyme pretreatment of FFPE tissue sections for proper staining Failure to pretreat FFPE tissue sections may result in reduced staining of
37. e di membrana e o citoplasmatiche Una colorazione citoplasmatica stata osservata anche in alcuni tumori a cellule squamose del polmone Risulta quindi importante analizzare l intensit della colorazione citoplasmatica in gliomi e carcinomi polmonari CARATTERISTICHE DELLA PERFORMANCE Specificit Nelle cellule A431 carcinoma epidermoide umano stimolate o non stimolate con EGF l espressione dell EGFr stata rilevata tramite anti EGFr di topo clone 31G7 Nelle analisi di trasferimento di proteine secondo il metodo di Western il clone 31G7 ha identificato una banda proteica da 170 kDa corrispondente al peso molecolare pubblicato dell EGFr Clone 31G7 non reagisce HER2 Clone 31G7 riconoscer EGFr vIII Riproducibilit Il Kit EGFr di Zymed stato testato su campioni tissutali tratti da 57 casi di carcinoma della testa e del collo Ciascun caso testato ha dimostrato l esistenza di una concordanza coerente nella riproducibilit nella valutazione della colorazione rispetto alla mancata colorazione Immunoreattivit Reattivit nei tessuti anomali In tutti gli studi menzionati qui di seguito si sono usati l anticorpo del clone 31G7 e tessuti fissati in formalina ed inclusi in paraffina Nei neoplasmi polmonari stata dimostrata una sovraespressione dell EGFr In uno studio condotto su 92 casi di tumore neuroendocrino il 28 dei casi di carcinoide tipico il 57 dei casi di carcinoide atipico il 42 dei casi
38. ecimen Incubate 30 min Rinse slides in fresh PBS Tween three times for 2 min each time Step 4 HRP polymer conjugate Blot excess liquid off slide Apply 2 drops 100 uL of HRP Goat anti Mouse IgG polymer conjugate Reagent 3 to each specimen Incubate 10 min Rinse slides in fresh PBS Tween three times 2 min each time Step 5 DAB Substrate Chromogen Solution See Section B3 for DAB preparation Blot excess liquid off slide Apply 2 drops 100 uL of DAB substrate chromogen Reagent 5 solution to each specimen Incubate 5 min Rinse under running tap water for 3 min Step 6 Counterstain Immerse slides in hematoxylin for 1 3 minutes Rinse slides under running tap water then immerse in either 0 25 dilute ammonia in water or 10 mM PBS pH 7 4 to blue the nuclei Note Counterstaining will produce a pale to dark blue coloration of the cell nuclei depending on the incubation time and potency of the hematoxylin Excessive or incomplete counterstaining may compromise proper interpretation of results Step 7 Coverslip Rinse slides briefly in distilled or deionized water Dehydrate slides in a graded series of ethanol 70 80 95 100 then clear once in xylene Non aqueous permanent mounting medium is recommended Apply 2 drops 100 uL of Histomount or Clearmount Mounting Solution to the slide then add coverslip QUALITY CONTROL Control Slides Zymed EGFr Kit cont
39. ell EGFr REAGENTI E MATERIALI OCCORRENTI MA NON ACCLUSI e Metanolo assoluto e Coprivetrino e Acqua distillata o deionizzata e Etanolo e Ematossilina di Mayer e Perossido di idrogeno al 30 e Microscopio ottico 1 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 e Mezzo di fissaggio quale Clearmount N Cat 00 8010 o Histomount N Cat 00 8030 e Soluzione fisiologica tamponata a base di fosfato Phosphate Buffer Saline PBS a 10 mM pH 7 4 con Tween 20 allo 0 05 e Controlli tissutali positivi e negativi Per le raccomandazioni specifiche relativamente alle fonti tissutali di controllo esterno pregasi consultare la sezione Controllo della qualit a pagina 4 e Bagnio vaschette per la colorazione e Temporizzatore e Xilene CONSERVAZIONE Conservare a 2 8 C Non usare dopo la data di scadenza Astenersi dall usare i reagenti qualora si rilevino dei segni evidenti di deterioramento o un calo di attivit sostanziale Di norma i reagenti acclusi al presente kit dovrebbero apparire quale liquido privo di materia particolata ISTRUZIONI PER L USO A Trattamento precolorazione delle sezioni tissutali Per una colorazione ottimale le sezioni tissutali devono essere pretrattate con una soluzione a base di Proteinase K Reagente 1 acclusa al Kit EGFR di Zymed La procedura in oggetto contempla il preriscaldamento della soluzione a base di Proteinase K a temperatura ambiente seguito dall incuba
40. en interpretar los resultados IHQ dentro del contexto del historial cl nico del paciente y de otras pruebas diagn sticas Principio del procedimiento El equipo Zymed EGFr utiliza el anticuerpo anti EGFr de rat n clon 31G7 de Zymed y la metodolog a de detecci n de polimerizado patentado por Zymed para detectar la presencia de anticuerpos primarios unidos a los ant genos en muestras fijadas en formalina y infiltradas en parafina Como parte de este equipo se proporcionan portas de control con estirpes celulares infiltradas en parafina y fijadas en formalina para demostrar la expresi n positiva y negativa del EGFr a fin de validar el rendimiento de la tinci n en cada an lisis IHQ REACTIVOS PROPORCIONADOS Suficientes para 35 pruebas Reactivo 1 Un frasco cuentagotas 8 mL de soluci n de proteinase K lista para usar Reactivo 2 Un frasco cuentagotas 4 mL de anticuerpo anti EGFr de rat n clon 31G7 listo para usar Reactivo 3 Un frasco cuentagotas 4 mL de suero de cabra anti IgG polimerizado de rat n conjugado con HRP listo para usar Reactivo 4 Un frasco cuentagotas 4 mL de IgG de rat n listo para usar para control negativo de isotipo Reactivo 5A Un frasco cuentagotas 1 mL de amortiguador concentrado 20X Reactivo 5B Un frasco cuentagotas 1 mL de crom geno DAB 20X Reactivo 5C Un frasco cuentagotas 1 mL de per xido de hidr geno al 0 6 20X Portas de control 5 portas de control sin tinci n Cada
41. epufferter Kochsalz Tween L sung waschen Schritt 5 DAB Substrat Chromogenl sung Die DAB Zubereitung ist in Abschnitt B3 beschrieben bersch ssige Fl ssigkeit vom Objekttr ger abtupfen Jeder Probe 2 Tropfen 100 uL DAB Substrat Chromogenl sung Reagenz 5 zugeben 5 Minuten inkubieren Unter flieBendem Leitungswasser 3 Minuten absp len Schritt 6 Gegenf rbung Die Objekttr ger 1 3 Minuten in H matoxylin eintauchen Die Objekttr ger unter flie endem Leitungswasser absp len und dann entweder in 0 25 verd nnten Ammoniak in Wasser oder in 10 mL phosphatgepufferte Kochsalzl sung pH 7 4 eintauchen um den Nukleus blau zu f rben Hinweis Bei der Gegenf rbung wird je nach Inkubationszeit und H matoxylinpotenz eine hell bis dunkelblaue F rbung des Zellkerns erzeugt Eine berm ige oder unvollst ndige Gegenf rbung beeintr chtigt die richtige Interpretation der Ergebnisse Schritt 7 Coverslip Die Objekttr ger kurz mit destilliertem oder entionisiertem Wasser absp len Dann die Objekttr ger stufenweise 70 96 80 95 100 96 in Ethanol dehydrieren und einmal in Xylen kl ren Es wird ein wasserfreies permanentes Fixativ empfohlen 2 Tropfen 100 uL Histomount oder Clearmount Fixativl sung auf den Objekttr ger geben und den Coverslip anbringen QUALITATSKONTROLLE Kontrollobjekttr ger Das EGFr Kit von Zymed enth lt 5 Kontrollobjekttr ger ungef rbt Jed
42. er Kontrollobjekttr ger enth lt 2 in Formalin fixierte in Paraffin eingebettete Zellreihenschnitte f r positive 2 und negative Levels der EGFr Proteinexpression F r jedes Farbungsverfahren sollte ein Kontrollobjekttr ger mitgefarbt werden um die Farbung zu validieren EGFr Control Slide Bild 1 EGFr Kontrollobjektt ger mit positiv Zellinie A und negativ Zellinie B Es ist zu beachten dass die Zelllinien nicht maBstabsgetreu dargestellt sind und kleiner als in dieser Zeichnung sind Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Kolorektalkarzinome eignen sich gut als positive Kontrollgewebe Kontroverse F rbungsergebnisse sollten dem Technical Service Department der Zymed Laboratories Inc gemeldet werden Das Zymed EGFr Kit ist nicht f r prognostische Informationen f r Patienten und oder Arzte vorgesehen und wurde auch nicht f r solche Zwecke validiert Die Objekttr ger sollten gem Tabelle 2 bewertet werden um die G ltigkeit jeder F rbung zu validieren Tabelle 2 Bewertung der Kontrollobjekttr ger und Kontrollobjekttr ger Reihenfolge der Bewertung Objekttr gerablesung 1 Kontrollobjekttr ger Eine positive braune Membranf rbung auf dem positiven mitgeliefert Kontrollobjekttr ger weist auf einen g ltigen Assay hin G ltigkeit jeder F rbung zu validieren Eine braune F rbung auf dem negativen Kontrollobjekttr ger weist auf eine nicht spezifische F rbung im Assay
43. ervarse como es de esperar para los controles positivos establecidos de tejido externo 3 Control negativo de tejido externo La ausencia de tinci n marr n espec fica en el control negativo de tejido confirma la falta de reactividad cruzada del equipo a los componentes celulares Si se produce la tinci n marr n espec fica en el control negativo de tejido los resultados obtenidos para la muestra del paciente deben ser considerados incorrectos 4 Tejido del paciente coloreado con control negativo del reactivo La ausencia de tinci n marr n espec fica indica la especificidad de la tinci n del ant geno blanco por el anticuerpo primario Otra tinci n marr n que se produzca en la muestra del paciente te ida con el control negativo del reactivo como en el tejido conectivo leucocitos eritrocitos o tejido necr tico debe ser considerada como tinci n de base inespec fica 5 Tejido del paciente coloreado con anticuerpo primario Cuando se detecta sobre expresi n del EGFr aparecer como tinci n marr n de la membrana con o sin tinci n del citoplasma de c lulas tumorales que han sido tratadas con anticuerpo primario INTERPRETACI N DE LA TINCI N Sistema de graduaci n Zymed EGFr La prote na del EGFr se expresa en una variedad de tejidos normales y neopl sicos El patr n de tinci n celular puede ser homog neo o heterog neo es decir tinci n de la membrana tinci n del citoplasma o
44. gsprotokoll Tabelle 1 Hinweis Keine durch Warmeeinwirkung induzierte Wiedergewinnung der Epitope HIER durchf hren Dadurch kann die EGFr Antigenit t vollkommen verloren gehen F rbungsverfahren Schritt 1 Peroxidase l schung Teil 30 iges Hydrogen Peroxid zu 9 Teilen reinem Methanol geben Gut mischen Die Objekttr ger f r 10 Min in Peroxidase l schung l sung tauchen Mit PBS 2 Min 3 mal waschen Mit Schritt 2 fortfahren Schritt 2 Enzym Digestion Die Proteinase K l sung Reagenz 1 auf Zimmertemperatur erw rmen Jeder Probe 2 Tropfen 100 uL vorgew rmtes Proteinase K zugeben F r 10 Minuten bei Zimmertemperatur inkubieren Die Objekttr ger dreimal f r jeweils 2 Minuten in frischer phosphatgepufferter Kochsalz Tween L sung waschen Schritt 3 Prim rantik rper oder negative Isotypkontrolle bersch ssige Fl ssigkeit vom Objekttr ger abtupfen Jeder Probe 2 Tropfen 100 uL Maus anti EGFr Reagenz 2 oder negative Isotypkontrolle Reagenz 4 zugeben 30 Minuten inkubieren Die Objekttr ger dreimal f r jeweils 2 Minuten in frischer phosphatgepufferter Kochsalz Tween L sung waschen Schritt 4 HRP Ziegen Anti Maus IgG Polymerkonjugat bersch ssige Fl ssigkeit vom Objekttr ger abtupfen Jeder Probe 2 Tropfen 100 uL HRP Ziegen Anti Maus IgG Polymerkonjugat Reagenz 3 zugeben 10 Minuten inkubieren Die Objekttr ger dreimal f r jeweils 2 Minuten in frischer phosphatg
45. h_support invitrogen com EGFr Kit Klon 31G7 SuperPicTure Detektionssystem Polymer F r immunohistochemische F rbung von epidermalem Wachstumsfaktor Rezeptor Epidermal Growth Factor Receptor EGFr Kat Nr 28 8763 Reicht fiir 35 Objekttr ger Tests VERWENDUNGSZWECK F r In Vitro Diagnostischem Gebrauch Die Interpretation muss im Zusammenhang mit der klinischen Vorgeschichte des Patienten und anderen Diagnostiktests eines qualifizierten Pathologen vorgenommen werden Beim EGFr Kit Klon 31G7 von Zymed handelt es sich um einen immunohistochemischen IHC Assay zur Identifizierung einer EGFr Proteinexpression in normalen und neoplastischen Geweben die zur histologischen Beurteilung in Formalin fixiert und in Paraffin eingebettet wurden Die test Ergebnisse miissen im Rahmen der klinischen Anamnese des Patienten und in Verbindung mit anderen diagnostischen Tests von qualifizierten Pathologen interpretiert werden Verfahrensprinzip Das EGFr Kit von Zymed verwendet den Zymed Mausantik rper Anti EGFr Klon 31G7 und Zymeds patentrechtlich geschiitzte SuperPicTure Detektionsmethode zur Erkennung der Gegenwart von den an Antigenen angelagerten prim ren Antik rpern in Formalin fixierten und in Paraffin eingebetteten Proben Die in Paraffin eingebetteten Gewebeschnitte m ssen deparaffiniert und rehydriert werden Kontrollobjekttr ger mit in Formalin fixierten in Paraffin eingebetteten Zellreihen die positive und negative EGFr
46. i Astenersi dal diluire ulteriormente i reagenti o dal sostituirli con reagenti provenienti da un diverso lotto di fabbricazione Si possono ottenere dei risultati falsi negativi a causa della degradazione graduale dell antigene nei tessuti I provini dovrebbero essere colorati entro 4 6 settimane dalla data di fissaggio sui vetrini se conservati in un luogo asciutto a temperatura ambiente 20 25 C Riferimenti 1 Marquez A et al Evaluation of Epidermal Growth Factor Receptor EGFR by Chromogenic in Situ Hybridization CISH and Immunohistochemistry IHC in Archival Gliomas Using Bright Field Microscopy Diagn Mol Pathol 13 1 1 8 2004 Arita N et al Epidermal Growth Factor Receptor in human glioma J Neurosurg 70 916 919 1989 Quezado MM et al Correlation of EGFR Gene Amplification and Overexpression by Chromogenic In Situ Hybridization CISH and Immunohistochemistry in High Grade Gliomas USCAP Annual Meeting Abstract 1334 2003 Piyathilake CJ et al Differential expression of growth factors in squamous cell carcinoma and precancerous lesions of the lung Clin Cancer Res 8 734 744 2002 Rappresentante Autorizzato MDSS Burckhardstr 1 D 30163 Hannover Germany Rappresentante Autorizzato per VDD 98 79 EC Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Z ZYMED Laboratories invitrogen immunodetection 561 Eccles Avenue South San Francisco CA 94080 Tel 800 955 6288 E Mail tec
47. idin to avoid the endogenous biotin activity avidin binding activity in tissue and cell samples that can cause background Any controversial staining should be reported to Zymed Laboratories Inc s Technical Service Department Zymed EGFr Kit is not intended to provide prognostic information to the patient and or physician and has not been validated for this purpose Slides should be evaluated according to Table 2 to verify the validity of each staining run Table 2 Control Slide amp Sample Slide Evaluation Slide Reading Order Evaluation 1 Control Slides Provided Presence of positive brown membrane staining on the positive control To verify the validity of the slide indicates a valid assay staining run Presence of brown staining on the negative control slide indicates that non specific staining occurred in the assay Assay results may be invalid due to over staining 2 External Positive Tissue Control Presence of brown membrane staining with or without cytoplasmic staining should be observed as expected for established external positive tissue controls 3 External Negative Tissue Control Absence of brown specific staining in the negative tissue control confirms the lack of kit cross reactivity to cellular components If brown specific staining occurs in the negative tissue control results obtained for the patient specimen should be considered invalid 4 Patient Tissue Stained With Negative
48. ingere di blu i nuclei Nota la controcolorazione dar adito ad una colorazione dei nuclei delle cellule la cui tonalit pu variare da azzurro chiaro a blu scuro a seconda del tempo di incubazione osservato e della potenza dell ematossilina impiegata Una colorazione eccessiva o incompleta potrebbe compromettere la correttezza dell interpretazione dei risultati Fase 7 Coprivetrino Risciacquare brevemente i vetrini in acqua distillata o deionizzata Disidratare i vetrini in una serie graduata di etanolo 70 80 95 100 quindi lavare immergendoli una volta in xilene Si raccomanda l impiego di un mezzo di fissaggio permanente non acquoso Applicare sul vetrino 2 gocce 100 uL della Soluzione fissativa Histomount o Clearmount quindi inserire nel coprivetrino CONTROLLO DELLA QUALITA Vetrini di controllo Il Kit EGFr di Zymed contiene 5 vetrini di controllo non colorati Ciascun vetrino contiene 2 sezioni di serie cellulari fissate in formalina ed incluse in paraffina rappresentanti livelli positivi 2 e negativi di espressione della proteina EGFr Si dovrebbe colorare un vetrino di controllo per ciascuna procedura di colorazione eseguita onde confermarne la validit Vetrini di controllo di EGFr Immagine 1 Vetrini di controllo di EGFr con la serie cellulari positiva 2 A e la serie cellulari negazione B Noti prego quello las series cellulari non sono disegnati alla scala e sia pi pic
49. ion has been observed in endometrial carcinoma where it correlates with myometrial invasion in a variety of lung neoplasia including metaplastic squamous epithelium squamous cell carcinoma adenocarcinoma and neuroendocrine lung tumors and in glioblastoma multiforme EGFr expression has also been reported in head and neck carcinoma and in correlation with advanced tumor size in invasive transitional cell carcinoma of the urinary bladder renal cell carcinoma and gastric carcinoma Variable expression of EGFr has been described in pilocytic low grade or anaplastic astrocytoma and in breast carcinoma frequently in combination with tumor metastasis In breast carcinoma EGFr expression does not demonstrate an inverse correlation with negative estrogen receptor ER status 1 1 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Principle of Procedure Zymed EGFr Kit utilizes Zymed Mouse anti EGFr clone 31G7 antibody and Zymed s proprietary SuperPicTure one step Polymer Detection methodology to detect the presence of primary antibodies bound to antigen in formalin fixed paraffin embedded specimens Paraffin embedded tissue sections require deparaffinization and rehydration then quenching of endogenous peroxidase by treatment with 3 hydrogen peroxidase in absolute methanol or with Zymed Peroxo Block Cat No 00 2015 After incubating the specimens with primary antibody Mouse anti EGFr clone 31G
50. irpe celular A y and negativo estirpe celular B Por favor anota que los estirpes celulares no son dibujado a escala y son m s pequefio que es indicado en la esquema El carcinoma de cabeza y cuello y o el carcinoma colorrectal humano puede ser til como fuente de tejido para control positivo Cualquier tinci n controvertida se debe comunicar al servicio t cnico de Zymed Laboratories Inc Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 El equipo Zymed EGFr no est dise ado para proporcionar informaci n diagn stica al paciente y o al m dico y no ha sido validado con este fin Los portaobjetos deben ser evaluados de acuerdo con la tabla 2 para verificar la validez de cada tinci n Tabla 2 Evaluaci n del portaobjetos de control y del portaobjetos de muestra Orden de lectura del portaobjetos Evaluaci n 1 Portaobjetos de control proporcionados La presencia de tinci n marr n positiva en la membrana del portaobjetos de control positivo indica que el an lisis es v lido Para verificar la validez de la e La presencia de tinci n marr n en el portaobjetos de control carrera de tinci n P p J negativo indica que en el an lisis se produjo una tinci n inespecifica Los resultados de los an lisis pueden ser incorrectos debido a la tinci n excesiva 2 Control positivo de tejido externo La presencia de tinci n marr n de la membrana con o sin tinci n del citoplasma debe obs
51. it importante sont visibles L apparence normale des r actifs dans ce kit est celle d un liquide exempt de particules MODE D EMPLOI A Traitement des sections de tissus avant la coloration Pour une coloration optimale les sections de tissus doivent tre pr trait es avec une solution de Proteinase K r actif n 1 tel que celle comprise dans le kit EGFr de Zymed Cette proc dure comprend un pr chauffage de la solution de proteinase K temp rature ambiante suivi d une incubation de la solution de proteinase K sur des sections de tissus pendant 10 minutes temp rature ambiante voir C3 tape n 1 de la proc dure de coloration page 4 pour des instructions suppl mentaires D autres traitements enzymatiques risquent de r duire ou d liminer compl tement l intensit de la coloration de l EGFr La restauration antig nique par la chaleur RAC ne peut pas tre utilis e avec l anti EGFr de souris clone 31G7 de Zymed B Pr paration du r actif Pr parer les r actifs suivants avant de colorer B1 Tampon de lavage non fourni solut tampon de phosphate PBS 10 mM pH 7 4 avec du Tween 20 0 05 B2 Solution de refroidissement la peroxydase non fournie H O 3 Ajouter 1 partie de peroxyde d hydrog ne 30 96 9 parties de m thanol absolu Bien m langer B3 Solution de substrat chromog ne DAB Pr parer apr s la tape n 3 du protocole la page 4 Les r actifs n 6A 6B et
52. kit sono acclusi dei vetrini di controllo contenenti delle serie cellulari fissate in formalina ed incluse in paraffina che presentano espressioni positive e negative di EGFr ai fini della convalida della performance di colorazione di ciascuna prova d immunocolorazione REAGENTI ACCLUSI Sufficienti per 35 test Reagente 1 Una boccetta contagocce da 8 mL di Soluzione a base di Proteinase K pronta per l uso Reagente 2 Una boccetta contagocce da 4 mL di Anticorpo anti EGFr di topo Clone 31G7 pronto per l uso Reagente 3 Una boccetta contagocce da 4 mL di Coniugato di polimere igG anti topo HRP capra pronto per l uso Reagente 4 Una boccetta contagocce da 4 mL di Controllo isotipo negativo jgg1 di topo pronto per l uso Reagente 5A Una boccetta contagocce da 1 mL di Concentrato di tampone 20X Reagente 5B Una boccetta contagocce da 1 mL di Cromogeno DAB 20X Reagente 5C Una boccetta contagocce da 1 mL di Perossido di idrogeno allo 0 696 20X Vetrini di controllo 5 vetrini di controllo non colorati Ciascun vetrino contiene 2 sezioni di serie cellulari fissate in formalina ed incluse in paraffina rappresentanti livelli positivi 2 e negativi di espressione della proteina EGFr Linee guida per la valutazione dell EGFr 1 set fotografici Sono acclusi un set di fotografie a colori di campioni di tessuto affetto da carcinoma del colon quali linee guida per l assegnazione di un punteggio ai vari livelli di espressione d
53. ladder A multivariate survival analysis Am J Clin Pathol 101 2 166 76 1994 21 Moscatello DK et al A naturally occurring mutant human epidermal growth factor receptor as a target for peptide vaccine immunotherapy of tumors Cancer Res 15 57 8 1419 24 1997 22 Marquez A et al Evaluation of Epidermal Growth Factor Receptor EGFR by Chromogenic in Situ Hybridization CISH and Immunohistochemistry IHC in Archival Gliomas Using Bright Field Microscopy Diagn Mol Pathol 13 1 1 8 2004 23 Arita N et al Epidermal Growth Factor Receptor in human glioma J Neurosurg 70 916 919 1989 24 Quezado MM et al Correlation of EGFR Gene Amplification and Overexpression by Chromogenic In Situ Hybridization CISH and Immunohistochemistry in High Grade Gliomas USCAP Annual Meeting Abstract 1334 2003 25 Piyathilake CJ et al Differential expression of growth factors in squamous cell carcinoma and precancerous lesions of the lung Clin Cancer Res 8 734 744 2002 9 Zymed Laboratories Inc 6 False positive results may be observed due to non immunological binding of proteins or substrate reaction products They may also be caused by pseudoperoxidase activity erythrocytes endogenous peroxidase activity cytochrome c or endogenous biotin liver breast brain kidney depending on the immunodetection method used 7 As in any immunohistochemical test a negative result means that the antigen was not detected not necessarily that the antigen was
54. le 4 Immunoreactivity Normal Tissues Tissue Type number tested Positive Staining 3 intensity rating system Adrenal gland 5 Cortical area granular cells 3 1 5 Negative 4 5 Bone marrow 3 Negative Breast 5 Lobular epithelial cells 2 Colon 5 Mucosal epithelial cells 1 1 5 Negative 4 5 Cerebellum 3 Molecular layer axons 3 2 3 2 1 3 Cerebrum 5 Negative Cervix 3 Squamous cells in basal layer 3 1 3 2 2 3 Endometrium 3 Negative Esophagus 4 Squamous cells in basal layer some submucosal glandular cells 2 3 4 Negative 1 4 Kidney 5 Tubules 1 4 5 Negative 1 5 Liver 5 Hepatocytes some bile duct 2 3 5 Negative 2 5 Lung 5 Negative Mesothelium 3 3 Muscle cardiac 5 Negative Muscle smooth 5 Negativei Muscle skeletal 5 Negative Nerve 3 Negative Ovary 3 Negative Pancreas 5 Pancreatic duct epithelium some acinic cells 1 2 5 Negative 3 5 Parathyroid 3 Negative Placenta 5 Syncytiotrophoblasts 3 4 5 2 1 5 Prostate 2 3 2 2 Skin 5 Basal layer some spinosum layer epithelial cells 3 2 5 1 1 5 Negative 2 5 Small intestine 3 Negative Spleen 5 Negative Stomach 5 Mucosal epithelial cells 1 3 5 Negative 2 5 Testis 5 Basemembrane 2 3 5 Negative 2 5 Thyroid gland 5 1 3 5 Negativ
55. mmunohistochemistry in High Grade Gliomas USCAP Annual Meeting Abstract 1334 2003 Piyathilake CJ et al Differential expression of growth factors in squamous cell carcinoma and precancerous lesions of the lung Clin Cancer Res 8 734 744 2002 Bevollm chtigter Repr sentant MDSS Burckhardstr 1 D 30163 Hannover Germany Bevollm chtigter Repr sentant f r IVDD 98 79 EC Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005
56. n beobachtet Klon 31G7 farbte 50 der Kopf und Hals Plattenepithelkarzinomproben einer Studie Variable EGFr Expression wurde bei Brustkarzonmen gemeldet trotz seiner Gegenwart an den Zellen die aus allen drei Keimbl ttern gewonnen wurden Eine Studie meldete EGFr berexpression in bis zu 35 96 aller Brustkrebse Klon 31G7 wurde auch in Studien der glandul ren Zellen von Endometrie und Prostatakarzinomen verwendet In einer Studie von 40 Fallen endometrioider Endometriekarzinome zeigte der Maus Anti EGFr Klon 31G7 bei 65 96 der Proben eine positive F rbung FEHLERBEHEBUNG M gliche Ursachen f r eine negative F rbung auf positiven Objekttr gern Die Reihenfolge der Schritte des F rbungsprotokolls wurde nicht eingehalten Die Inkubationsschritte f r den prim ren oder sekund ren Antik rper wurden ausgelassen Labile Antigene wurden zerst rt Die Probe wurde nicht richtig fixiert und oder verarbeitet Die Probe trocknete w hrend der Farbung aus DD M gliche Ursachen f r eine schwache F rbung auf allen Objekttr gern 1 Nach der Sp lung blieb zuviel Fl ssigkeit in der Probe zur ck 3 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 2 3 4 Die Inkubationszeiten waren nicht ausreichend Das Substrat wurde falsch vorbereitet Unvollst ndige Deparaffinierung F rbung ist von einer starken Hintergrundf rbung begleitet M gliche Ursachen f r starke Hintergrundf rbung CY ME ROUES EX
57. n appropri e Si les sections tissulaires FFIP ne sont pas pr trait es cela risque d entra ner une coloration r duite de faux r sultats n gatifs 2 Les r actifs ont t optimis s Ne pas diluer davantage ni remplacer les r actifs par des lots de fabrication diff rente 3 De faux r sultats n gatifs peuvent galement tre obtenus suite une d gradation de l antig ne dans les tissus au fil du temps Les sp cimens doivent tre color s dans les 4 6 semaines apr s avoir t attach s aux lames lorsqu ils sont conserv s au sec temp rature ambiante 20 25 C R f rences 1 Marquez A et al Evaluation of Epidermal Growth Factor Receptor EGFR by Chromogenic in Situ Hybridization CISH and Immunohistochemistry IHC in Archival Gliomas Using Bright Field Microscopy Diagn Mol Pathol 13 1 1 8 2004 2 Arita N et al Epidermal Growth Factor Receptor in human glioma J Neurosurg 70 916 919 1989 3 Quezado MM et al Correlation of EGFR Gene Amplification and Overexpression by Chromogenic In Situ Hybridization CISH and Immunohistochemistry in High Grade Gliomas USCAP Annual Meeting Abstract 1334 2003 4 Piyathilake CJ et al Differential expression of growth factors in squamous cell carcinoma and precancerous lesions of the lung Clin Cancer Res 8 734 744 2002 Repr sentant Autoris MDSS Burckhardstr 1 D 30163 Hannover Germany Repr sentant Autoris pour IVDD 98 79 EC Zymed La
58. n colon carcinoma tissue is included as a guideline to scoring EGFr expression REAGENTS amp MATERIALS REQUIRED BUT NOT PROVIDED e Absolute methanol Coverslips Distilled or deionized water Ethanol Mayer s hematoxylin 30 Hydrogen peroxide Light microscope Mounting media such as Clearmount Cat No 00 8010 or Histomount Cat No 00 8030 10 mM phosphate buffered saline PBS pH 7 4 with 0 05 Tween 20 Positive and negative tissue controls See Quality Control page 4 for specific recommendations on external control tissue sources Staining jars or baths e Timer e Xylene STORAGE Store at 2 8 C Do not use after expiration date Reagents should not be used if deterioration or substantial loss of activity is evident The normal appearance of the reagents in this kit is a liquid free of particulate matter 2 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 INSTRUCTIONS FOR USE A Specimen Preparation Paraffin Embedded Sections 10 neutral buffered formalin fixative is a suitable fixative for most antigens of clinical significance Tissues that have been fixed in 10 neutral buffered formalin prior to paraffin embedding are suitable for use with Mouse anti EGFr clone 31G7 Tissue sections must be deparaffinized and rehydrated before staining see C1 Staining Procedure for instructions Pre Treatment of Tissue Sections Prior to Staining For optimal staining tissue sections require pret
59. nsformation of various cell types from a benign to a malignant phenotype EGFF is expressed in many normal epithelial tissues particularly in the basal layers of stratified or pseudostratified epithelium and in squamous epithelium In the normal tissues examined Zymed s Mouse anti EGFr antibody clone 31G7 stains epidermal cells of the skin colon testis mesothelium kidney placenta and prostate Positive staining appears as a linear to finely granular pattern within the cell membrane and adjacent cytoplasm or as coarsely granular cytoplasmic staining Zymed s 31G7 antibody reacts with EGFr and does not recognize the highly homologous c erB 2 HER 2 protein Western blotting analysis with an EGFr vIII transfected NIH 3T3 cell line confirmed that clone 31G7 recognizes the 145 kDa variant III form of EGFr in addition to recognizing the wild type form of EGFr EGFr expression has been reported in the basal layer of the skin epidermis hair follicles ductal and myoepithelial cells of the breast ductal and acinar cells of the pancreas glandular cells of the endometrium and prostate and basal cells of the epididymis The distribution of EGF receptors suggests a role for epidermal growth factor in the control of cellular proliferation and the differentiation of surface epithelia Many types of neoplastic tissues exhibit abnormal expression of EGFr Over expression of EGFr as the result of gene amplification and or increased protein transcript
60. nstrate positive staining results obtained for the test specimens should be considered invalid Negative Tissue Control Use an external negative tissue control known to be negative for EGFr staining that has been fixed processed and embedded in a manner identical to the patient samples in each staining run This will verify the specificity of the IHC primary antibody for identification of the target antigen and provide a demonstration of specific background staining false positive staining The wide variety of cell types present in most tissue sections may also be used as internal negative control sites to verify the test s performance specifications If specific staining false positive staining occurs in the negative tissue control results obtained for the patient specimens should be considered invalid Nonspecific Negative Reagent Control Use a non specific Negative Isotype Control Reagent 4 in place of the primary antibody with a section of each patient specimen to evaluate nonspecific staining and to allow clear interpretation of specific staining at the antigen site The incubation period for the Negative Isotype Control should correspond to that of the primary antibody To differentiate endogenous enzyme activity from specific immunoreactivity additional patient tissues may be stained exclusively with substrate chromogen EGFr kit utilizes SuperPicTure a polymer detection method without the use of biotin avidin or streptav
61. nt Cat No 00 8010 is recommended C Staining Procedure All reagents should be equilibrated to room temperature 20 25 C prior to immunostaining unless otherwise specified C1 Deparaffinization and Rehydration 1 Immerse slides in xylene twice for 5 min each time 2 Immerse slides in 100 ethanol twice for 2 min each time 3 Immerse slides in 95 ethanol for 2 min 4 Immerse slides in 80 ethanol for 2 min then proceed to Peroxidase Quenching C2 Immunohistostaining Protocol Table 1 Note Do not perform heat induced epitope retrieval HIER as it may result in complete loss of EGFr antigenicity 3 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Table 1 Staining Procedure Step 1 Peroxidase Quenching For paraffin embedded tissues add 1 part of 30 hydrogen peroxide to 9 parts of absolute methanol Mix well b Submerge slides in Peroxidase Quenching Solution for 10 minutes c Wash with deionized water 2 min 3 times Step 2 Enzyme Digestion Step 3 Primary Antibody or Negative Isotype Control Pre warm the proteinase K solution Reagent 1 to room temperature Apply 2 drops 100 uL of pre warmed proteinase K to each specimen Incubate for 10 min at room temperature Rinse slides in fresh PBS Tween three times for 2 min each time Blot excess buffer off slide Apply 2 drops 100 uL of Mouse anti EGFr Reagent 2 or Negative Isotype Control Reagent 4 to each sp
62. o o sin diluir Mezclar bien b Ba ar los portaobjetos de la soluci n para la eliminaci n de la Peroxidasa durante 10 minutos c Lavar con agua desionizada 2 minutos 3 veces Precalentar la soluci n de proteinase K reactivo 1 a temperatura ambiente Aplicar 2 gotas 100 uL de proteinase K precalentada a cada muestra Incubar durante 10 minutos a temperatura ambiente Enjuagar los portas en PBS Tween fresco tres veces durante 2 minutos cada vez Paso 3 Anticuerpo primario o control negativo de isotipo Eliminar el exceso de amortiguador del porta Aplicar 2 gotas 100 uL de anticuerpo anti EGFr de rat n reactivo 2 o control negativo de isotipo reactivo 4 a cada muestra Incubar durante 30 minutos Enjuagar los portas en PBS Tween fresco tres veces durante 2 minutos cada vez Paso 4 Conjugado de Pol merio HRP Eliminar el exceso de l quido del porta Aplicar 2 gotas 100 uL de suero de cabra anti IgG polimerizado de rat n conjugado con HRP reactivo 3 a cada muestra Incubar durante 10 minutos Enjuagar los portas en PBS Tween fresco tres veces durante 2 minutos cada vez Paso 5 Soluci n sustrato crom geno DAB consulte la secci n B3 para la preparaci n del DAB Eliminar el exceso de l quido del porta Aplicar 2 gotas 100 uL de soluci n sustrato crom geno DAB reactivo 5 a cada muestra Incubar durante 5 minutos Enjuagar con agua corriente durante 3 minutos Paso 6
63. or in astrocytic tumours is specifically associated with glioblastoma multiforme Virchows Arch Pathol Anat 420 321 325 1992 12 Christensen ME The EGF receptor system in head and neck carcinomas and normal tissues Immunohistochemical and quantitative studies Dan Med Bull 45 2 121 34 1998 13 NealDE et al Epidermal growth factor receptors in human bladder cancer comparison of invasive and superficial tumors Lancet 1 366 368 1985 14 Sainsbury JRC et al Epidermal growth factor receptors and estrogen receptors in human breast cancer Lancet 1 364 366 1985 15 Toi M et al Immunocytochemical and biochemical analysis of epidermal growth factor receptor expression in human breast cancer tissues relationship to estrogen receptor and lymphatic invasion Int J Cancer 43 220 225 1989 16 Veale D et al Epidermal growth factor receptors in non small cell lung cancer Brit J Cancer 55 513 516 1987 17 Berger MS et al Epidermal growth factor receptors in lung tumors J Pathol 152 297 307 1987 18 Patridge M et al Expression of epidermal growth factor receptor on oral squamous cell carcinoma Brit J Oral Maxillofac Surg 26 381 389 1988 19 Sartor CI et al Role of epidermal growth factor receptor and STAT 3 activation in autonomous proliferation of SUM 102PT human breast cancer cells Cancer Res 57 5 978 87 1997 20 Nguyen PL et al Expression of epidermal growth factor receptor in invasive transitional cell carcinoma of the urinary b
64. ouris clone 31G7 a manifest une coloration positive dans 65 des sp cimens D PANNAGE Causes possibles d une coloration n gative sur des lames positives Les tapes du protocole de coloration n ont pas t ex cut es dans le bon ordre Les tapes d incubation des anticorps primaires ou secondaires ont t omises Les antig nes labiles ont t d truites Le sp cimen tait mal attach et ou trait Le sp cimen s est d sydrat pendant la coloration GA doo ES ES Causes possibles pour une coloration faible sur toutes les lames 1 Le sp cimen a retenu un exc s de liquide apr s les tapes de rin age 2 Les temps d incubation n taient pas suffisants 3 Le substrat tait mal pr par 4 La d paraffination tait incompl te la coloration peut tre accompagn e d une coloration de fond lev e Causes possibles pour une coloration de fond lev e 1 L activit de la peroxydase endog ne n tait pas compl tement bloqu e 2 La d paraffination tait incompl te 3 Application excessive de l adh sif tissulaire 4 Mauvais rin age des lames 5 Surd veloppement du substrat 6 D shydratation du sp cimen pendant la coloration 5 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 LIMITATIONS SPECIFIQUES AU PRODUIT 1 L anti EGFr de souris clone 31G7 de Zymed n cessite un pr traitement enzymatique des sections tissulaires FFIP pour obtenir une coloratio
65. paration du DAB ponger l exc s de liquide sur la lame Appliquer 2 gouttes 100 uL de solution substrat chromog ne DAB r actif n 5 sur chaque sp cimen Incuber pendant 5 min Rincer sous l eau du robinet pendant 3 min tape n 6 Coloration de contraste Plonger les lames dans de l h matoxyline pendant 1 3 minutes Rincer les lames sous l eau du robinet les plonger ensuite soit dans de l ammoniac 0 25 dilu dans de l eau soit dans du PBS 10 mM pH 7 4 pour faire bleuir les noyaux Remarque la coloration de contraste produira une coloration des noyaux des cellules de bleu clair bleu fonc en fonction du temps d incubation et de la puissance de l h matoxyline Une coloration de contraste excessive ou incompl te risque de compromettre une interpr tation exacte des r sultats tape n 7 Lamelle couvre objet Rincer bri vement les lames dans de l eau distill e ou d sionis e D shydrater les lames dans une s rie class e d thanol 70 96 80 95 96 100 et nettoyer ensuite une fois dans du xyl ne Un moyen de fixation permanent non aqueux est recommand Appliquer 2 gouttes 100 uL de solution de fixation Histomount ou Clearmount sur la lame et ajouter la lamelle couvre objet CONTROLE DE LA QUALITE Lames de contr le Le kit EGFr de Zymed comprend 5 lames de contr le non color es Chaque lame contient 2 sections de lign es cellul
66. r tampon 20 x R actif n 5B Un flacon compte gouttes 1 mL de chromog ne DAB 20 x R actif n 5C Un flacon compte gouttes 1 mL de peroxyde d hydrog ne 0 6 20 x Lames de contr le 5 lames de contr le non color es Chaque lame contient 2 sections de lign es cellulaires fix es dans du formol et incorpor es dans de la paraffine repr sentant des niveaux d expression de prot ine EGFr positifs 2 et n gatifs Directives pour l valuation d EGFr 1 jeu de photos Un jeu de photos en couleur des sp cimens du cancer du c lon R ACTIFS ET MAT RIEL N CESSAIRES MAIS NON FOURNIS e M thanol absolu Lamelles couvre objet Eau distill e ou d sionis e thanol H matoxyline de Mayer Peroxyde d hydrog ne 30 96 Microscope optique Support pour pr parations microscopiques tel que Clearmount No de cat 00 8010 ou Histomount No de cat 00 8030 e Solut tampon de phosphate PBS 10 mM pH 7 4 avec du Tween 20 0 05 96 1 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 e Contr les tissulaires positifs ou n gatifs voir Contr le de la qualit page 4 pour des recommandations sp cifiques sur les sources de tissus de contr le externe e Bocaux ou bains de coloration e Minuterie e Xyl ne STOCKAGE Conserver entre 2 et 8 C Ne pas utiliser apr s la date de p remption Les r actifs ne doivent pas tre utilis s si des signes de d gradation ou d une perte d activ
67. r e de la membranaire est Mod r 2 Mod r observ e dans des cellules tumorales Une coloration intense de la membrane est observ e Intense 3 Intense dans des cellules tumorales On observe habituellement la souillure membranous d EGFr dans diverses tumeurs telles que la t te et le cou le sein l intestin du c lon et l ovaire Cependant on a observ la souillure membranous et cytoplasmique seul ou en association dans le glioma On a galement observ la souillure cytoplasmique dans quelques cancers 4 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 squamous de cellules du poumon Par cons quent il peut tre important de rapporter l intensit de la souillure cytoplasmique dans le glioma et des carcinomas du poumon CARACTERISTIQUES DE PERFORMANCE Sp cificit Une expression d EGFr dans des cellules A431 cancer pidermoide humain soit stimul soit non stimul avec de EGF a t d tect e par l anti EGFr de souris clone 31G7 Lors d une analyse d immunotransfert le clone 31G7 a reconnu une bande prot ique 170 kDa correspondant au poids mol culaire publi de EGFr Clone 31G7 ne r agit pas avec HER2 Clone 31G7 reconna tra EGFr vIII Reproductibilit Le kit EGFr de Zymed a t test sur 57 cas de sp cimens de tissus de cancers de la t te et du cou Chaque cas test manifestait un accord constant dans la reproductibilit en ce qui concerne l valuation de la colo
68. ra Un mL de soluci n sustrato crom geno DAB es suficiente para diez cortes tisulares B4 Contra tinci n no se proporciona con el equipo La hematoxilina de Mayer Cat N 00 8001 es la recomendada para la contra tinci n B5 Soluci n de montaje no se proporciona con el equipo Se recomienda el medio de montaje permanente y no acuoso como Histomount Cat N 00 8030 o Clearmount Cat N 00 8010 C Procedimiento de tinci n Todos los reactivos deben equilibrarse a temperatura ambiente 20 25 C antes de proceder a la inmunotinci n excepto indicaci n en contrario C1 Desparafinizaci n y rehidrataci n 1 Sumergir los portaobjetos en xileno dos veces durante 5 minutos cada vez 2 Sumergir los portaobjetos en etanol al 100 dos veces durante 2 minutos cada vez 3 Sumergir los portaobjetos en etanol al 95 durante 2 minutos 4 Sumergir los portas en etanol al 80 durante 2 minutos y luego proceder a la extinci n de la peroxidasa C2 Protocolo de inmunotinci n histol gica tabla 1 Nota No realizar la recuperaci n del ep topo inducida por calor Heat Induced Epitope Retrieval HIER ya que puede dar lugar a la p rdida completa de la antigenicidad al EGFr 2 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 Procedimiento de tinci n Paso 1 Paso 2 Digesti n enzim tica Para tejidos en parafina afiadir 1 parte de per xido de hidr geno al 30 a nueve partes de metanol pur
69. raron un resultado de 2 a 4 en la tinci n para EGFr En otro estudio se observ sobre expresi n del EGFr en el 5696 de las muestras de adenocarcinoma pulmonar y el 8446 de las muestras de carcinoma pulmonar de las c lulas escamosas En un estudio el clon 31G7 tifi el 50 de las muestras de carcinoma de las c lulas escamosas de cabeza y cuello Se inform de la expresi n variable de EGFr en muestras de carcinoma de mama a pesar de su presencia en c lulas derivadas de los tres planos germinales un estudio inform de la sobre expresi n del EGFr hasta en un 35 de los c nceres de mama El clon 31G7 tambi n ha sido utilizado en estudios de c lulas glandulares de carcinoma endometrial y prost tico En un estudio de 40 casos de carcinoma endometrial endometrioide el anticuerpo anti EGFr de rat n clon 31G7 mostr tinci n positiva en el 6596 de las muestras DETECCI N Y DIAGN STICO DE AVER AS Causas posibles de la tinci n negativa en portaobjetos positivos Los pasos en el protocolo de tinci n se realizaron en una secuencia incorrecta Se omitieron los pasos de incubaci n del anticuerpo primario o secundario Se destruyeron ant genos l biles La muestra no se fij y o proces correctamente La muestra se deshidrat durante la tinci n posce ae Causas posibles de la tinci n d bil en todos los portaobjetos 1 La muestra retuvo exceso de l quido despu s de los aclarados 2 Los tiempos de incubaci n no fueron s
70. ration par rapport a pas de coloration Immunor activit R activit tissulaire anormale Toutes les tudes cit es ci dessous ont utilis l anticorps anti clone 31G7 et des tissus fix s dans du formol et incorpor s dans de la paraffine La surexpression d EGFr a t d montr e dans des n oplasmes pulmonaires Dans une tude effectu e sur 92 tumeurs neuro endocriniennes 28 de carcino des typiques 57 de carcino des atypiques 42 de cancers neuro endocriniens grandes cellules 39 de cancers neuro endocriniens petites et grandes cellules et 22 de cancers du poumon petites cellules ont donn un score de 2 4 pour la coloration d EGFr Dans une autre tude une surexpression de l EGFr fut observ e dans 56 des sp cimens d ad nocarcinomes du poumon et 84 des sp cimens des cancers spino cellulaires du poumon Dans une tude le clone 31G7 a color 50 des sp cimens de carcinomes spino cellulaires de la t te et du cou Une expression d EGFr variable a t report e dans des sp cimens de cancer du sein malgr sa pr sence sur des cellules d riv es des trois feuillets embryonnaires une tude a rapport une surexpression de l EGFr dans jusqu 35 des cancers du sein Le clone 31G7 a galement t utilis dans des tudes de cellules glandulaires de cancers de l ut rus et de la prostate Dans une tude de 40 cas de carcinomes entom tro des de l ut rus l anti EGFr de s
71. reatment with proteinase K solution Reagent 1 as included in the Zymed EGFr Kit Clone 31G7 This procedure includes pre warming the proteinase K solution to room temperature followed by incubation of the proteinase K solution on tissue sections for 10 minutes at room temperature see C3 Step 1 of Staining Procedure page 4 for instructions Other enzyme treatments may reduce or eliminate the intensity of EGFr staining altogether Heat induced epitope retrieval HIER CANNOT be used with Zymed s Mouse anti EGFr clone 31G7 B Reagent Preparation Prepare the following reagents prior to staining B1 Washing Buffer Not provided 10 mM Phosphate Buffered Saline PBS pH 7 4 with 0 05 Tween 20 B2 Peroxidase Quenching Solution Not provided 3 H O Add 1 part of 30 hydrogen peroxide to 9 parts of absolute methanol Mix well B3 DAB Substrate Chromogen Solution Prepare after step 3 of the protocol on page 4 Reagents 5A 5B and 5C are 20X concentrate reagents Add 1 drop Reagent 5A 1 drop Reagent 5B and 1 drop Reagent 5C to 1 mL distilled or deionized water Mix well Protect from light and use within one hour One mL of DAB substrate chromogen solution is sufficient for ten tissue sections B4 Counterstain Not provided Mayer s hematoxylin Cat No 00 8001 is recommended for counterstaining B5 Mounting Solution Not provided Non aqueous permanent mounting medium such as Histomount Cat No 00 8030 or Clearmou
72. rexpression No staining in tumor cells None 0 Negative Faint or barely perceptible membrane Very Weak to Weak 1 Weak staining is observed Partial staining in part of the membrane or weak complete membrane staining is observed Moderate membrane staining is observed Moderate 2 Moderate in tumor cells Strong membrane staining is observed in Strong 3 Strong tumor cells EGFr membrane staining is usually observed in various tumors such as head and neck breast colon and ovary However membrane and cytoplasmic staining has been observed either alone or in combination in glioma Cytoplasmic staining has also been observed in some squamous cell cancers of the lung Therefore it may be important to report the intensity of cytoplasmic staining in glioma and lung carcinomas PERFORMANCE CHARACTERISTICS Specificity EGFr expression in A431 human epidermoid carcinoma cells either stimulated or non stimulated with EGF has been detected by Mouse anti EGFr clone 31G7 On Western blot analysis clone 31G7 recognized a 170 kDa protein band corresponding to the published molecular weight of EGFr Clone 31G7 does not react with HER2 Clone 31G7 will recognize EGFr vIII Reproducibility Zymed EGFr Kit was tested on 57 cases of colon carcinoma tissues specimens Each tested case demonstrated consistent agreement in reproducibility in the evaluation of staining versus no staining Immunoreactivity
73. s Lungenfliigels Folglich kann es wichtig sein ber die Intensit t des cytoplasmatisch Befleckens Glioma und Lungenfl gel in den carcinomas zu berichten LEISTUNGSMERKMALE Spezifit t EGFr Expression in A431 humanen Epidermoidkarzinom Zellen mit oder ohne EGF Stimulation wurde von dem Maus Anti EGFr Klon 31G7 entdeckt Bei der Western Blotanalyse erkannte Klon 31G7 ein 170 kDa Proteinband das dem ver ffentlichten Molekulargewicht von EGFr entspricht EGFr erkennt nicht mit HER2 Clone 31G7 erkennt mit EGFr vIII Reproduzierbarkeit Das EGFr Kit von Zymed wurde in 57 Fallen von Kopf und Halzkarzinomgewebeproben gepriift Bei jedem Testfall zeigte sich bereinstimmende Reproduzierbarkeit der Bewertung von F rbungen im Vergleich zu Nichtf rbungen Immunreaktivit t Abnormale Gewebereaktivit t In allen unten aufgef hrten Studien wurden Klon 31G7 Antik rper und in Formalin fixierte in Paraffin eingebettete Gewebe verwendet EGFr Uberexpression wurde in pulmonalen Neoplasmen nachgewiesen In einer Studie von 92 neuroendokrinen Tumoren wurde mit 28 typisches Karzinoid 57 atypisches Karzinoid 42 gro zelliges neuroendokrines Karzinom 39 Mischung aus klein und gro zelligen neuroendokrinen Karzinomen und 22 kleinzelliges Lungenkarzinom eine Bewertung von 2 bis 4 EGFr F rbung nachgewiesen In einer anderen Studie wurde EGFr Uberexpression in 56 Lungen Adenokarzinom und 84 Lungen Plattenepithelkarzinomprobe
74. uficientes 3 El sustrato no se prepar correctamente 4 La desparafinizaci n fue incompleta la tinci n puede acompa arse por un fondo elevado Causas posibles para la tinci n elevada del fondo 1 La actividad de la peroxidasa end gena no se bloque completamente 2 La desparafinizaci n fue incompleta 3 Aplicaci n excesiva del adhesivo tisular 4 Aclarado inadecuado de los portaobjetos 5 Excesivo desarrollo del sustrato 6 Deshidrataci n de la muestra durante la tinci n 5 Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 LIMITACIONES CONCRETAS DEL PRODUCTO 1 El anti EGFr de rat n clon 31G7 de Zymed requiere un pretratamiento enzim tico de los cortes tisulares FFPE para una tinci n adecuada La imposibilidad del pretratamiento de los cortes tisulares FFPE puede dar lugar a la tinci n reducida de resultados negativos falsos Los reactivos han sido optimizados No diluir ni reemplazar reactivos de distintos lotes de fabricaci n Debido a la degradaci n del ant geno en los tejidos a lo largo del tiempo se pueden obtener resultados negativos falsos Las muestras deben ser sometidas a tinci n entre las 4 y 6 semanas siguientes a su montaje en los portaobjetos cuando se almacenan a una temperatura ambiente seca 20 25 C Referencias l Marquez A et al Evaluation of Epidermal Growth Factor Receptor EGFR by Chromogenic in Situ Hybridization CISH and Immunohistochemistry IHC in
75. ve or negative staining should be complemented by morphological studies using proper positive and negative internal and external controls as well as other diagnostic tests It is the responsibility of a qualified pathologist who is familiar with the proper use of IHC antibodies reagents and methods to assess all of the steps used to prepare and interpret the final IHC preparation Any deviation from recommended test procedures may invalidate declared expected results appropriate controls must be employed and documented Reagents may demonstrate unexpected reactions in previously untested tissues The possibility of unexpected reactions even in tested tissue groups cannot be completely eliminated due to biological variability of antigen expression in pathological tissues Contact Zymed s Technical Service Department at Tel 1 650 871 4494 Fax 1 650 871 4499 or e mail tech zymed com with documented unexpected reactions Zymed Laboratories Inc Cat No 28 8763 PIN 30925 4 6 2005 RELATED PRODUCTS Table 5 Related Products Product Clone Cat No Mouse anti EGFr Concentrate 31G7 28 0005 Mouse anti EGFr 2 Gen Predilute 31G7 08 1205 Mouse anti EGFr V Line Predilute 31G7 08 4205 EGFr Amplification Probe forCISH 84 1300 SuperPicTure Polymer Kit Broad Spectrum DAB 87 9663 REFERENCES 1 Cohen S et al A native 170 000 epidermal growth factor receptor kinase complex from shed plasma membrane vesicles J Biol Chem
76. zione della medesima sulle sezioni tissutali per 10 minuti a temperatura ambiente per le istruzioni pregasi consultare la sezione C3 Fase 1 della Procedura di colorazione a pagina 4 Altri trattamenti enzimatici potrebbero attenuare l intensit della colorazione dell EGFr se non eliminarla del tutto Con l anti EGFr di topo clone 31G7 di Zymed NON SI PU usare il richiamo dei determinanti antigeni indotto mediante calore Heat Induced Epitope Retrieval HIER B Preparazione dei reagenti Prima della colorazione preparare i seguenti reagenti B1 Tampone di lavaggio non accluso soluzione fisiologica tamponata a base di fosfato Phosphate Buffer Saline PBS a 10 mM pH 7 4 con Tween 20 allo 0 05 B2 Soluzione per il quenching di perossidasi non acclusa H O al 3 Aggiungere 1 parte di perossido di idrogeno al 30 per ogni 9 parti di metanolo assoluto Miscelare accuratamente B3 Soluzione di substrato cromogeno DAB preparare dopo aver eseguito la Fase 3 del protocollo a pagina 4 I reagenti 6A 6B e 6C sono forniti in forma concentrata 20X Aggiungere 1 goccia del Reagente 6A 1 goccia del Reagente 6B ed 1 goccia del Reagente 6C in 1 mL di acqua distillata o deionizzata Miscelare accuratamente Conservare lontano da fonti di luce ed usare entro un ora Un mL di Soluzione di substrato cromogeno DAB sufficiente per dieci sezioni tissutali B4 Controcolorazione non acclusa Per la controcolorazione si raccomanda l

EGFr Kit (Clone 31G7) - Thermo Fisher Scientific

Contents

Download Pdf Manuals

Related Search

Related Contents