Chapter 21
Contents
1. Other 150 IHC STAINING METHODS FIFTH EDITION Troubleshooting Endogenous Blocks Background staining is defined as unexpected or undesirable staining seen on the test or control tissue which does not represent the target antigen Frequent causes of background staining are endogenous enzyme activity and endogenous biotin Peroxidase is an enzyme of the oxido reductase class that reacts with a substrate containing hydrogen peroxide as the electron acceptor To block this activity a variety of hydrogen peroxide reagents can be applied to cells producing this enzyme Alkaline phosphatase is an enzyme having various isoforms which are produced in the leukocytes liver bone intestine placenta and Regan carcinoma Addition of levamisole to the chromogen substrate will inhibit endogenous alkaline phosphatase activity with the exception of the intestinal isoform If necessary this can be blocked with a weak acid wash such as 0 03 0 5 N HCI Biotin a B vitamin may be protein bound to tissue and can interfere with proper interpretation of staining patterns when using a streptavidin or avidin reagent To block this binding a biotin avidin block Peroxidase block 3 H O Methanol H O Sodium azide Peroxidase Block S2001 Other Alkaline Phosphatase block Levamisole 0 03 N HCI not for use on cryostat tissue Other Biotin block Biotin Block2. mesangial and endothelial cells of the renal glomerulus as well as pancreatic acinar cells 1 2 In the human eye the antibody labels the pigmented posterior and the anterior epithelia of the human iris including the muscle portion dilator pupillae of the anterior epithelium as well as the nonpigmented and pigmented ciliary epithelia 4 In the ciliary epithelia vimentin was coexpressed with cytokeratin 4 Abnormal tissues The antibody labeled 17 20 sarcomas 16 18 melanomas 4 4 meningeomas and 3 3 schwannomas and was the sole intermediate filament present in these tumours In addition variable percentages 10 to 57 percent of carcinomas neuroendocrine carcinomas neuroblastomas thymomas and mesotheliomas were positive with the antibody With the exception of the neuroblastomas cytokeratin was coexpressed with vimentin in these tumours Among adenocarcinomas more than 50 percent of papillary carcinomas of the thyroid as well as renal endometrial ovarian and lung carcinomas were labeled by the antibody and coexpressed keratins and vimentin Negative Control Tissue Performance characteristics Normal tissues Skeletal and cardiac muscle cells epidermal squamous urothelial colonic and gastric mucosal and glial cells as well as neurons are consistently negative with the antibody Comments CLIA 2003 Sec 493 1273 3 Mandates that fluorescent and immunohistochemical stains must be check
3. chromogen Reduce incubation time Specification reagent Sheet Substrate chromogen reagent prepared incorrectly Repeat incubation with correctly prepared chromogen reagent Specification Sheet Secondary or link antibody cross reacts with antigens Absorb link antibody with tissue protein extract or species specific normal serum from 51 60 from tissue specimen tissue donor Secondary or link antibody and or tertiary reagents too Repeat staining Determine correct concentration for each reagent Incubation 11 13 concentrated temperature and incubation time will affect results To determine optimal incubation protocol vary both the time and temperature for each reagent in the IHC staining protocol Slides inadequately rinsed Gently rinse slide with wash buffer bottle and place in wash bath for 5 minutes Gentle 5 6 agitation of the wash bath may increase effectiveness when used with cytoplasmic or nuclear staining protocols Insufficient saline or detergent in wash buffer High sensitivity staining systems may require higher concentrations of saline or 115 121 detergent in the wash buffer Refer to the staining system specification sheet for optimal formulation Blocking serum or wrong blocking serum used Block with serum from the host of the secondary or link antibody Avoid serum that 115 121 contains auto immune immunoglobulins Alternatively a serum free protein block acking immunoglobulins may be substituted for the serum block Sections incorre
4. 1 28 Immunoreactivity diminished or destroyed during Use a paraffin wax with a melting temperature 55 58 C Wax used for embedding 29 33 embedding process should not exceed 60 C Immunoreactivity diminished or destroyed during Oven temperature not to exceed 60 C 29 33 dewaxing at high oven temperature NOTE The intensity of immunostaining may be diminished when tissue is exposed to prolonged heat Refer to the primary antibody specification sheet for additional information Immunoreactivity diminished or destroyed on pre cut The intensity of immunostaining may be diminished when pre cut tissue sections are 29 33 tissue sections exposed to air Use freshly cut sections and reseal paraffin embedded blocks Immunoreactivity diminished or destroyed by the More common on frozen sections apply the primary antibody prior to the enzymatic 29 33 115 enzyme blocking reagent altering a specific epitope block to insure its reaction In such cases the blocking reagent can be applied at any point after the primary and before the enzyme labeled components Excessive wash buffer or blocking serum remaining on Excess reagent will dilute the next consecutive reagent Repeat staining making sure to 11 13 tissue section prior to application of IHC reagents wipe away excess washing buffer and blocking serum Demasking protocol is inappropriate or has been Some tissue antigens require proteolytic enzyme digestion or heat induced antigen 2053905 omitted Rep
5. Chapter 21 Troubleshooting Karen N Atwood and Dako Technical Support Group Immunohistochemistry is a multi step process that requires specialized training in the processing of tissue the selection of appropriate reagents and interpretation of the stained tissue sections In general IHC staining techniques allow for the visualization of antigens by sequential application of a specific antibody to the antigen a secondary antibody to the primary antibody an enzyme complex and a chromogenic substrate The enzymatic activation of the chromogen results in a visible reaction product at the antigen site Because of its highly complex nature the causes of unexpected negative reactions undesired specific staining or undesired background could be difficult to isolate The information contained in this chapter should enable you to rapidly pinpoint and resolve problems encountered during the staining procedure Section One is a compilation of common problems encountered when using immunohistochemical staining reagents the underlying causes of staining failure and recommended corrective actions The chart is divided into sections describing little or no staining general background staining and limited background staining Section Two presents a method of systematically adding one IHC reagent at a time to determine at which stage non specific or undesired staining may be occurring in a peroxidase streptavidin biotin staining system Sect
6. X0590 Other Protein block Protein Block X0909 Normal sera from host species of the secondary antibody Other IHC STAINING METHODS FIFTH EDITION 151 Troubleshooting Section Four Using a Typical Specification Sheet for an IVD Antibody Information You Need to Know Information Located on the Specification Sheet Regulatory Status of the Intended use Indicates that a product meets the FDA requirements as a Primary Antibody For in vitro diagnostic use clinical diagnostic product Likewise a CE icon indicates the reagent meets European Union requirements Patient test results do not require an FDA disclaimer Tissue Preparation Specimen preparation Indicates the type of specimen that was used during Paraffin sections The antibody can be used for labeling paraffin embedded tissue sections fixed in formalin Pre treatment of tissues with heat induced epitope retrieval is required Optimal results are obtained with 10 mmol L citrate buffer pH 6 0 Less optimal results are obtained with 10 mmol L Tris buffer 1 mmol L EDTA pH 9 0 The tissue sections should not dry out during the treatment or during the following immunocytochemical staining procedure Frozen sections and cell preparations The antibody can be used for labeling frozen sections or fixed cell smears validation studies In many cases this would include formalin fixed tissue and frozen sections Use of other fixat
7. atic sections Negative Control Tissue Perform complete staining protocol SLIDE 5 NO STAINING SEEN GO TO NEXT STEP v 148 IHC STAINING METHODS FIFTH EDITION Red Blue color observed on Negative Control Tissue Monoclonal antibody Possible contamination Polyclonal antibody Possible contamination or undesired antibody in the host Ig fraction Antigen retrieval lipofusion artifact may appear as granule staining in liver and cardiac tissue or as specific staining in pancreatic sections Troubleshooting Negative Control Reagent Reagent Result Action Red Blue color observed Negative Control Reagent ee Human tissue Perform the peroxidase blocking protocol from Slide Perform complete staining protocol 2 under Background Staining Encountered with HRP Peroxidase Reagents Perform a biotin block if required protein block if required apply the appropriate negative reagent control see below apply biotinylated secondary antibody apply streptavidin HRP reagent and DAB Prepare a negative reagent control Polyclonal non immunized sera from the same species diluted to the same protein concentration as the primary antibody Monoclonal negative reagent control that matches the isotype as the primary antibody Additionally the diluent used to manufacture a monoclonal primary antibody and isotypic negative control should contain the sam
8. ctly dewaxed Prepare new sections and deparaffinize according to standard laboratory protocol using 115 121 fresh xylene or xylene substitute Non specific binding of the secondary antibody with an Use a secondary antibody that has been absorbed against a species specimen or use 51 60 animal tissue specimen a secondary antibody produced in a host that exhibits little or no cross reactivity with See also the tissue source 115 121 Instrument malfunction Ensure automated stainer is programmed correctly and is running to manufacturer s Specification Specification Sheet 140 IHC STAINING METHODS FIFTH EDITION Troubleshooting Specimen tissue and negative reagent control slides show background staining Positive and negative control tissue show appropriate specific staining May involve several tissue elements such as connective tissue adipose tissue and epithelium Specimen held for too long in a cross linking fixative Standardize routine fixation Proteolytic digestion or antigen retrieval will break down 29 33 usually in formalin causing masking of antigenic cross linking and render some tissue antigens reactive Refer to the primary antibody determinants due to aldehydes cross linking and specification sheet for additional information increased hydrophobicity of tissue Sectioned portion of specimen not penetrated by Fix tissue biopsy for longer period of time or fix smaller pieces to ensure compl
9. dase methodologies Repeat staining Commercial phosphate buffers may contain additives that will inhibit alkaline phosphates activity Avoid sodium azide in diluents and buffers A concentration of 15mM L sodium azide which is routinely added to IHC reagents to inhibit bacterial growth will not impair HRP conjugated labels 138 IHC STAINING METHODS FIFTH EDITION Troubleshooting Possible Cause Solution Antigen levels are too low for detection by the Utilize a higher sensitivity staining system 57 60 employed visualization system May be due to loss Prolong incubation time of primary antibody n MEE aligrentiation msome dumo or loss af Re optimize incubation times and concentrations of ancillary reagents antigenicity due to sub optimal tissue fixation Perform antigen retrieval if applicable using a range of pH buffers Steric hindrance due to high antigen level and possible Re optimize concentration of the primary antibody and ancillary reagents Antibody 51 60 prozone effect concentration of the primary antibody may be too high Use of inappropriate fixative Check manufacturer s specifications regarding recommended fixative 29 33 Use of certain fixatives may damage or destroy antigens or epitopes in the tissue specimen Use of non cross linking fixatives may allow the elution of antigens soluble in IHC reagents Different fixatives may affect standardization 29 33 see also 2
10. e ions Diluents containing sodium or phosphate ions may change the sensitivity of some monoclonal antibodies Calculation g or total protein concentration of primary antibody divided by dilution factor of primary antibody x g or total protein concentration of negative reagent control divided by x dilution factor of negative reagent control IHC STAINING METHODS FIFTH EDITION 149 Troubleshooting Section Three Tissue Specimen Tissue Specimen Successful staining of tissue with an IHC marker is dependent on the type and preparation of the specimen Record in the chart below the species of the animal to be tested the tissue source or organ from which it was collected the collection method how the specimen was fixed and tissue Species Organ tissue source Collection Surgical specimen biopsy Post mortem specimen Fine needle aspirate Peripheral blood include anti coagulant Brushing Biologic fluid Cell culture Other Tissue preparation Paraffin embedded Plastic embedded Cryostat section Cytospin Cell smear Mono layer cultured cells Other Tissue fixation Type of fixative Length of time Size of specimen Tissue mounting Slide mount Tissue thickness Gelatin glue commercial adhesive or starch in the water bath
11. eated reuse of antigen retrieval buffer retrieval performed prior to staining The need for pretreatment depends on the type and extent of fixation specific characteristics of the antigen and the type of antibody used Use the pretreatment method recommended by the manufacturer No single pretreatment is suitable for all applications Do not reuse buffer Specification Sheet Sections incorrectly dewaxed Prepare new sections and deparaffinize according to standard laboratory protocol 115 121 using fresh xylene or xylene substitute Failure to achieve the optimal temperature required for When using a waterbath or steamer allow sufficient time for the retrieval buffer to 51 65 heat induced antigen retrieval equilibrate to a temperature range of 95 99 C Athigh altitude greater than 4 500 feet the buffer will boil at less than 95 C Utilize a closed heating system such as a pressure cooker autoclave or Pascal or utilize a low temperature protocol if standardization of the validated procedure is not affected Excessive or incomplete counterstaining Re optimize concentration of counterstain and incubation time 51 65 Instrument malfunction Ensure automated stainer is programmed correctly and is running to manufacturer s Specification specifications Sheet IHC STAINING METHODS FIFTH EDITION 139 Troubleshooting Positive control tissue shows adequate specific staining with little or no background stain
12. ed for appropriate positive and negative reactivity each time they are used Most IVD antibody specification sheet will list tissue that will exhibit positive and negative staining patterns in the Performance Characteristics section NOTE abnormal tissue will not necessarily be labeled Both negative and positive tissue controls should be processed using the same fixation embedding mounting drying epitope retrieval and immunostaining protocols as the patient tissue IHC STAINING METHODS FIFTH EDITION 153 Troubleshooting References 1 Wood G et al Suppression of Endogenous Avidin Binding Activity in Tissues and Its Relevance to Biotin Avidin Detection Systems Journal of Histochemistry and Cytochemistry 2981 29 1196 204 2 Sayaki H et al Azure B as a Counterstain in the Immunohistological Evaluation of Heavily Pigmented Nevomelanocytic Lesions Applied Immunohistochemistry 1995 3 268 71 Federal Register January 24 2003 68 42CFR Part 493 College of American Pathology Anatomic Pathology Checklist October 2005 154 IHC STAINING METHODS FIFTH EDITION
13. egative reagent control serum 127 130 For polyclonal antibodies dilute the negative reagent control serum until the protein concentration is equal to that of the primary antibody For monoclonal antibodies dilute the negative reagent control serum until the Ig concentration is equal to that of the primary antibody Contaminating antibodies in the negative control serum Replace the negative reagent control serum repeat staining protocol 127 130 are cross reacting with proteins from the specimen tissue Negative reagent control serum contaminated with Replace product with non contaminated serum 8 9 bacterial or fungal growth Limited Background Areas of inconsistent staining on controls specimens and glass slides Protein trapped beneath the tissue during the mounting process will allow partial lifting of the section Pooling of IHC reagents beneath the section or partial detachment of the tissue from the slide may occur Avoid the use of commercial adhesives glue starch or gelatin in water baths when mounting tissue sections Avoid allowing water from an initial section mounting to flow over an area where additional sections will be mounted This is particularly important when using charged or silanized slides 51 65 115 121 Undissolved granules of chromogen Insure that chromogen in tablet or powder form is completely dissolved or switch to a 115 121 liquid chromogen Incomplete removal of embedding med
14. els of pigment exist in the tissue the red chromogen may be partially obscured Since bleaching protocols to remove melanin may compromise tissue antigenicity it should be avoided if at all possible ne Brown Red color observed B Positive Control Tissue Indicates endogenous peroxidase activity in the tissue sections It is zi DAB AEC Counterstain ES i o present in all hemoprotein containing tissue including erythrocytes muscle liver kidney granulocytes and monocytes NO STAINING SEEN GO TO NEXT STEP Block with three percent hydrogen peroxide or other peroxidase Y blocking reagent Using a new bottle of hydrogen peroxide perform a three percent H 0 peroxidase block followed by DAB and an appropriate counterstain M Positive Control Tissue Brown Red color observed 4 P B Peroxidase Block Secondary Antibody Indicates endogenous biotin activity in the tissue sections Protein 3 Streptavidin HRP Ma pz DAB AEC Counterstain bound biotin may be found in adrenal liver kidney Gl tract lung spleen brain mammary gland adipose tissue lymphoid tissue and cell grown in culture media containing biotin RPMI NO STAINING SEEN GO TO NEXT STEP v NCTC MEME Block with a biotin block or switch to a staining system that is not dependent on the streptavidin biotin reaction IHC STAINING METHODS FIFTH EDITION 145 Troubleshooting Reagents Positive Control Tissue w Pero
15. en pancreas brain mammary gland adipose tissue lymphoid tissue and cells grown in culture media containing biotin as a nutrient IHC STAINING METHODS FIFTH EDITION 143 Troubleshooting Background of skeletal or smooth muscle tissue in positive control tissue negative control tissue specimen tissue and negative reagent control Possible Cause Solution See Page Cause is not understood It is possibly due to Should not interfere with interpretation of specific staining 115 121 antibodies to muscle antigens in primary and negative reagent control serum Undesired Specific Staining Positive staining of leucocyte membranes in specimen tissue positive control negative tissue control and negative reagent control Possible Cause Solution See Page Binding of the F portion of Ig by F receptors on the cell Use F ab or F ab fragments for the primary and secondary antibodies rather than intact 115 121 membrane of macrophages monocytes granulocytes antibodies and some lymphocytes Add detergent to the wash buffer Positive staining of histiocytes and granulocytes in the specimen tissue only with a marker not normally reactive with these cells Possible Cause Solution See Page Phagocytosis of antigens may render phagocytes Rare Should not interfere with interpretation of specific staining 115 121 positive for the same Positive membrane staining of specimen tissue and negative reagent c
16. ete 29 33 fixative Loss of antigenicity in unfixed tissue Unfixed penetration tissue tends to bind all reagents nonspecifically Sectioned portion contains crush artifact caused Serum proteins diffuse through tissue and are fixed in place 115 121 by grossing tissue with dull scalpel or razor Serum Re cut tissue using sharp blade proteins diffuse through tissue and are fixed in place Sectioned portion of specimen contains necrotic or Ignore physically damaged portions of stained tissue sections 115 121 otherwise damaged elements Excessive or unevenly applied subbing agent on poly Some IHC reagents may bind to these products resulting in a light stain over the entire 115 121 L lysine charged or silanized slides slide surface Some slides may be unevenly coated and will exhibit the above problems on only a portion of the tissue or glass Antigen diffusion prior to fixation causing specific Avoid delays in fixation of the tissue 115 121 background outside the expected antigen site Tissue sections too thick Cut tissue sections thinner Formalin fixed paraffin embedded tissue sections should 29 33 be approximately 4 6 um cryostat section pm IHC STAINING METHODS FIFTH EDITION 141 Troubleshooting Negative reagent control slide shows background Positive control tissue negative control tissue and specimen tissue show expected specific staining Negative control serum insufficiently diluted Use properly diluted n
17. idase staining system Unquenched endogenous peroxidase activity may Use alternate or prolonged peroxidase blocks or use another enzyme label such as alkaline 115 121 be seen in all hemoprotein containing specimens phosphatase including hemoglobin in erythrocytes myoglobin Eosinophils and mast cells are particularly resistant to peroxidase quenching Use a in muscle cells cytochrome in granulocytes and peroxidase blocker monocytes and catalases in liver and kidney Use special stains eosin will stain eosinophils a bright red orange Background seen in all control and specimen tissue when using an alkaline phosphatase staining system Possible Cause Solution See Page Unquenched endogenous alkaline phosphatase activity Add levamisole to the alkaline phosphatase chromogen reagent or use another enzyme 115 121 may be seen in leucocytes kidney liver bone ovary label such as horseradish peroxidase Intestinal alkaline phosphatase is not quenched bladder salivary glands placenta and gastro intestinal by the addition of levamisole Pretreat the tissue with 0 03 N HCI tissue Background seen in all control and specimen tissue when using a biotin streptavidin staining system Endogenous protein bound biotin water soluble B Use a biotin block or chose another non biotin based staining system 115 121 vitamin High amounts of biotin are found in adrenal liver and kidney Lesser amounts are found in the Gl tract lung sple
18. ing Specimen tissue shows little or no specific staining with variable background staining of several tissue elements Specimen held for too long in a cross linking fixative Standardize routine fixation Proteolytic digestion or antigen retrieval will break down 51 65 usually in formalin causing masking of antigenic cross linking and render some tissue antigens reactive Refer to the primary antibody determinants due to aldehydes cross linking and specification sheet for additional information increased hydrophobicity of tissue Serum proteins diffuse through tissue and are fixed in place 115 121 Re cut tissue using sharp blade Sectioned portion contains crush artifact caused by grossing tissue with dull scalpel or razor Sectioned portion of specimen contains necrotic or Ignore physically damaged portions of stained tissue sections 51 65 otherwise damaged elements Section portion of specimen not penetrated by fixative Fix tissue biopsy for longer period of time or fix smaller pieces to ensure complete 29 33 Loss of antigenicity in unfixed tissue penetration Unfixed tissue tends to bind all reagents nonspecifically 115 121 see also 51 56 General Background Background seen in all control tissue and specimen tissue May see marked background staining in several tissue elements such as connective tissue adipose tissue and epithelium Excessive incubation with substrate
19. ion Three is a simple chart used to define the type of tissue specimen the IHC staining and ancillary reagents already in place in the laboratory and the staining protocol used by the laboratory personnel You are encouraged to copy this chart and use it to help troubleshoot any problems you may encounter with your staining systems Section Four is a guide to reading a manufacturers specification sheet for IVD antibodies This includes general information for use in immunohistochemistry including fixation recommended visualization systems recommended titer and diluent pretreatment and selection of required controls IHC STAINING METHODS FIFTH EDITION 137 Troubleshooting Section One Inadequate Staining Little or no staining of controls or specimen tissue except for counterstain May show little or no background staining Primary antibody or labeled reagent omitted Reagent Repeat the procedure using the manufacturer s staining system specification sheet or 57 60 used in wrong order standard operating procedure reagent checklist as established by the individual laboratory Excessively diluted or excessively concentrated Determine correct concentration for each reagent Depending on the degree of staining 7 11 12 reagents inappropriate incubation time and obtained if any a 2 to 5 fold change in concentration may be needed Incubation temperature temperature and incubation time are inver
20. ium Remove embedding medium thoroughly using fresh reagents 115 121 Incomplete dezenkerization of tissue fixed with B5 or Perform dezenkerization with fresh reagents 29 33 mercury containing reagents Bacterial or yeast contamination from mounting Clean and refill waterbath 115 121 waterbath Partial drying of tissue prior to fixation Unaffected Immerse tissue promptly in fixative or holding reagent 115 121 areas show normal staining Keep moist during the entire staining process Use a humidity or moist chamber during incubation steps When using an automated staining instrument addition of wet towels to the sink may prevent drying of slides Instrument malfunction Ensure automated stainer is programmed correctly and is running to manufacturer s Specification Specification Sheet Adipose or connective tissue in specimen negative control tissue positive control tissue and negative reagent control slides Background in connective and epithelial tissue Hydrophobic and ionic interactions between Nonspecific staining of fatty tissue rarely interferes with interpretation of specific 115 121 immunoglobulins and lipoid substances in fatty tissue staining and can usually be disregarded Primary antibody and negative reagent control serum 11 13 are insufficiently diluted Reoptimize the dilution of the primary antibody and negative control serum De zenk dezenkerization is the use of iodine to rem
21. ives requires validation by each individual laboratory This section also indicates the optimal epitope retrieval procedure and warns against procedures that may destroy the epitope Specimen preparation and staining procedure sections can and will change periodically to reflect changes in technology So remember to retain copies of each version of the reagent specification sheet Version numbers are usually found on each page Choosing the Visualization System Staining procedure Visualization This antibody can be used with an immunperoxidase staining method Follow the procedure enclosed with the selected visualization kit Automation The antibody is well suited for immunocytochemical staining using automated platforms Indicates the recommended visualization system to be used with the antibody It also indicates that the antibody can be used for automated staining NOTE If your state regulatory agency requires written documentation that a reagent can be used for automated staining and this indication is not listed on the specification sheet you may wish to contact the manufacturer s technical support group for further information Diluting the Primary Antibody Staining procedure Dilution Monoclonal Mouse Anti Vimentin may be used at a dilution range of 1 50 1 100 when applied on formalin fixed paraffin embedded sections of human tonsil Includes a suggested dilution range for the antibody and the recomme
22. nded diluent The dilution range is merely a suggested starting point for an individual laboratory Optimal conditions may vary depending on specimen preparation method temperature of the laboratory or automated instrumentation Negative Reagent Control Reagent provided Isotype IgG1 kappa Staining procedure The recommended negative control is Mouse monoclonal IgG1 diluted to the same mouse IgG concentration as the primary antibody Positive and negative controls should be run simultaneously with patient specimen Use of a negative reagent control is required by the College of American Pathologists CAP based on Clinical Laboratory Improvement Amendments CLIA 2003 for each patient or patient block in a staining run 152 IHC STAINING METHODS FIFTH EDITION Troubleshooting Information You Need to Know Positive Control Tissue Information Located on the Specification Sheet Performance characteristics Normal tissues In general most human mesenchymal cells are labeled by the antibody including fibrocytes lipocytes smooth muscle cells vascular endothelial cells astrocytes peripheral nerve Schwann cells macrophages including Kupffer cells as well as myoepithelial cells of sweat and salivary glands and of breast which are all labeled strongly Also positive with variable intensity and distribution are the follicular cells of the thyroid adrenal cortex renal distal tubules and
23. ontrol tissue when using a horseradish peroxidase staining system Tissue from persons infected with Hepatitis B virus Utilize a non peroxidase staining system 115 121 and expressing Hepatitis B surface antigen may exhibit undesired staining Miscellaneous Loss of viability of cell cultures Possible Cause Solution See Page Some manufacturers produce antibodies and reagents Utilize an in vivo product for application on viable cells 115 121 for in vitro use only These products may contain For use on cell cultures only sodium azide may be dialyzed out of some reagents preservatives usually sodium azide which is a Contact Dako Technical Support for additional information known poison 144 IHC STAINING METHODS FIFTH EDITION Troubleshooting Section Two Troubleshooting flow chart Use this flow chart to determine source s of non specific staining when using an immunohistochemical protocol Background Staining Encountered with HRP Peroxidase Reagents Reagents Result Action Brown endogenous pigment such as melanin observed Positive Control Tissue To distinguish melanin pigment from DAB chromogen Azure B can Counterstain with hematoxylin i i na be used as a counterstain The melanin stains blue green while the DAB remains brown NO STAINING SEEN GO TO NEXT STEP Analternate method is to use AEC as the chromogen However if v high lev
24. ove mercury pigment 142 IHC STAINING METHODS FIFTH EDITION Troubleshooting Epithelial tissue in specimen negative control tissue positive control tissue and negative reagent control slides Staining is moderate to marked especially in epidermal epithelium Background in epithelia accompanies background in connective tissue Both the primary antibody and negative control Usea higher dilution of the primary antibody and negative control serum oa WIZ serum contain contaminating antibodies to epithelial Increase the incubation time elements possibly cytokeratins Replace the antibody Excessive formalin fixation of tissues may Proteolytic digestion or antigen retrieval will break down cross linking and render some 29 33 increase protein cross linking resulting in tissue tissue antigens reactive Refer to the primary antibody and or the negative reagent 115 121 hydrophobicity control specification sheet for appropriate pretreatment Focal cytoplasmic staining observed in epithelium in the specimen tissue Possible Cause Solution See Page Focal cytoplasmic staining is seen particularly in This observation is rare and should not interfere with interpretation of specific staining 115 121 intermediate and superficial layers of the epidermis May be caused by passive absorption of plasma proteins into degenerating epidermal cells Background seen in all control and specimen tissue when using an immunoperox
25. s package inserts and reagent labels Dissociation of primary antibody during washing or A feature of low affinity antibodies 5 6 incubation with link antibodies Polyclonal primary antiserum Attempt staining at low dilutions Monoclonal primary antibody Replace with higher affinity antibody of identical specificity Re optimize incubation times for washing buffer and link antibody Use of alcohol based counterstain and or alcohol Repeat staining using water based counterstain and mounting media 16 17 based mounting media with aqueous based chromogens Use a permanent chromogen such as DAB DAB that is not affected by organic solvents Excessive counterstaining may compromise proper Use a counterstain that 115 121 interpretation of results Will not excessively stain tissue sections Can be diluted so as not to obliterate the specific signal Reduce incubation time of the counterstain Incorrect preparation of substrate chromogen mixture Repeat substrate chromogen treatment with correctly prepared reagent Specification Staining intensity is decreased when excess DAB DAB is present in the working reagent Sheet Incompatible buffer used for preparation of enzyme and Check compatibility of buffer ingredients with enzyme and substrate chromogen reagents 51 60 substrate chromogen reagents Use of PBS wash buffer with an alkaline phosphatase staining system Sodium azide in reagent diluent or buffer baths for immunoperoxi
26. sely proportional and will affect results To determine optimal incubation protocol vary either the time or temperature for each reagent in the IHC staining system Generally incubation times can be extended if little or no background was detected Primary antibody diluted with inappropriate buffer Check formula and compatibility of antibody diluent A change of ion content and or pH 51 60 Use of PBS or TBS as an antibody diluent Lack of of the antibody diluent can cause a diminution in the sensitivity of the antibody Addition stabilizing or carrier protein Detergent in diluent of NaCl should be avoided This problem is primarily seen with monoclonal antibodies Primary antibody defective one or several secondary Replace defective or expired antibody repeat staining protocol replacing one reagent at T 8 or ancillary reagents defective Do NOT use product atime with fresh in date reagents after expiration date stamped on via Store products according to each product specification sheet or package insert f using a neat or concentrated antibody and directed by the manufacturer to store frozen it may be aliquoted to avoid repeated freezing and thawing For freezing use a 70 C to 80 C freezer or a non frost free 20 C freezer A frost free freezer will cycle on and off which will cause damage to the antibody Do not freeze ready to use or customer diluted products Follow manufacturer recommendations on product specification sheet
27. staining system that is not dependent on the streptavidin biotin reaction a Positive Control Tissue Red Blue color observed Se M ui Biotin Block if required Secondary Indicates non specific or undesired binding of the secondary Antibody Streptavidin AP Fast Red ibod ME his mn e Fuchsin or BCIP NBT Counterstain antibody to the tissue sections This primarily occurs when the secondary antiserum has not been prepared for use on a specific species tissue NO STAINING SEEN GO TO NEXT STEP T To determine if this is the problem absorb out non specific proteins by adding 2 5 or 10 uL of normal serum from the species of tissue to be stained per 100 uL of the secondary antibody IHC STAINING METHODS FIFTH EDITION 147 Troubleshooting Reagent Positive Control Tissue Biotin Block if required Negative a Reagent Control Secondary Antibody Zz Streptavidin AP Fast Red Fuchsin or BCIP NBT Counterstain NO STAINING SEEN GO TO NEXT STEP v Result Action Red Blue color observed May indicate non specific binding of the primary antibody carrier protein Perform a protein block with normal serum from the host of the link antibody or a protein block add 0 05 0 1 TWEEN 20 to wash buffer to decrease protein attachment Antigen retrieval lipofusion artifact may appear as granule staining in liver and cardiac tissue or as specific staining in pancre
28. ved on Negative Control Tissue Monoclonal antibody Possible contamination Polyclonal antibody Possible contamination or undesired antibody in the host Ig fraction Antigen retrieval lipofusion artifact may appear as granule staining in liver and cardiac tissue or as specific staining in pancreatic sections Troubleshooting Background Staining Encountered with Alkaline Phosphatase Reagents Result Action Red Blue color observed X Positive Control Tissue 5 Fast Red Fuchsin or BCIP NBT Indicates endogenous alkaline phosphatase activity in the tissue a Counterstain sections It is present in liver kidney GI tract bone bladder ovary salivary gland placenta leukemic necrotic or degenerated cells NO STAINING SEEN GO TO NEXT STEP Block with levamisole Intestinal alkaline phosphatase may be hd quenched by the addition of 0 03 N HCI prior to the addition of the alkaline phosphatase u f Red Blue color observed b Positive Control Tissue B Streptavidin AP Fast Red Fuchsin Indicates endogenous biotin activity in the tissue sections Protein a or BCIP NBT Counterstain bound biotin may be found in adrenal liver kidney Gl tract lung spleen brain mammary gland adipose tissue lymphoid tissue and cells grown in culture media containing biotin RPMI NO STAINING SEEN GO TO NEXT STEP v NCTC MEME Block with a biotin block or switch to a
29. xidase Block Biotin Block if required E Secondary Antibody Streptavidin HRP DAB AEC Counterstain Result Action NO STAINING SEEN GO TO NEXT STEP v Positive Control Tissue Peroxidase Block Biotin Block if required Negative Reagent Control Secondary Antibody Streptavidin HRP DAB AEC SLIDE 5 NO STAINING SEEN GO TO NEXT STEP v Negative Control Tissue Perform complete staining protocol SLIDE 6 NO STAINING SEEN GO TO NEXT STEP v 146 IHC STAINING METHODS FIFTH EDITION Brown Red color observed Indicates non specific or undesired binding of the secondary antibody to the tissue sections This primarily occurs when the secondary antiserum has not been prepared for use on a specific species tissue To determine if this is the problem absorb out non specific proteins by adding 2 5 or 10 uL of normal serum from the species of tissue to be stained per 100 uL of the secondary antibody Brown Red color observed May indicate non specific binding of the primary antibody carrier protein Perform a protein block with normal serum from the host of the link antibody add 0 05 0 1 TWEEN 20 to wash buffer to decrease protein attachment Antigen retrieval lipofusion artifact may appear as granule staining in liver and cardiac tissue or as specific staining in pancreatic sections Brown Red color obser
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