User Manual - ENZ-51038-K040 - ProteoStat Amyloid Plaque
Contents
1. dehyde solution Dilute 0 4 gram paraformaldehyde to a final volume of 10 mL with 1X Assay Buffer Mix well ProteoStat Amyloid Detection Reagent For optimal staining the concentration of the ProteoStat Amyloid dye will vary depending upon the application and must be deter mined by the end user The dilution suggested and described in the following paragraph serves only as a guideline Modifications may be required depending upon the particular specimen type employed in the application Dilute a sufficient amount of the ProteoStat Amyloid Detection Reagent for the number of samples to be assayed as follows For every 2 mL of 1X Assay Buffer from section A1 above add 1 uL of ProteoStat Amyloid Detection Reagent Mix well B STAINING TiSSUE SECTIONS 1 Specimen Preparation and Pretreatment Tissue sections should be about 5 um thick Frozen tissue sec tions should be mounted on positively charged glass slides and fixed with cold acetone The embedded tissue sections should be fixed in formalin immediately after excision and embedded in par affin Tissue sections should be mounted on positively charged glass slides a Paraffin embedded Tissue Sections Paraffin embedded tissue sections should be deparaffinized prior to staining Bake the mi croscope slide mounted specimens at 60 80 C for 5 15 minutes 3 to melt the paraffin Then the slides are immersed in a series of Coplin jars containing the following solutions2. Enzo Enabling Discovery in Life Science ProteoStat Amyloid Plaque Detection Kit for fluorescence microscopy Instruction Manual Cat No ENZ 51038 K040 For research use only Rev 1 0 October 2010 Notice to Purchaser The ProteoStat Amyloid Plaque Detection Kit is a member of the CELLestial product line reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for cell analysis applications CELLestial reagents and kits are optimal for use in demanding cell analysis applications involving confocal microscopy flow cytometry microplate readers and HCS HTS where consistency and reproducibility are required This product is manufactured and sold by ENZO LIFE SCIENCES INC for research use only by the end user in the research market and is not intended for diagnostic or therapeutic use Purchase does not include any right or license to use develop or otherwise exploit this product commercially Any commercial use development or exploitation of this product or development using this product without the express prior written authorization of ENZO LIFE SCIENCES INC is strictly prohibited Limited Warranty These products are offered under a limited warranty The products are guaranteed to meet appropriate specifications described in the package insert at the time of shipment Enzo Life Sciences sole obligation is to replace the product to the extent of the purchase price All
3. for the times indicated or until the paraffin is completely solubilized Soak Number Reagent Duration of Soak 1 Xylenes 10 minutes 2 Xylenes 2 minutes NOTE Use fresh reagents in each jar for each batch of slides and dis card in appropriate waste containers when finished Xylenes saturate rapidly with paraffin Use fresh xylenes for each batch of 5 slides b Frozen Tissues Sections Frozen tissue should be thawed at room temperature Wash the specimens deparaffinized and frozen sequentially in a series of Coplin jars containing the following solutions for the times indicated Soak Number Reagent Duration of Soak 3 100 alcohol 1 minute 4 100 alcohol 1 minute 5 90 alcohol 1 minute 6 70 alcohol 1 minute 7 50 alcohol 1 minute 8 Deionized Water 1 minute Carefully remove excess water dispense 200 uL of 4 formalde hyde from section V A2 page 3 per 1 cm surface of tissue for 15 min at 37 C After fixing wash the tissue sections in deionized water Carefully remove excess water and and dispense 200 uL of ProteoStat Detection Reagent see section V A3 page 3 per 1 cm surface to cover the tissue sections Protect samples from light and incubate for 3 minutes at room temperature Rinse the tissue sections in water and de stain in 196 acetic acid for 20 min 8 10 Finally wash the de stained tissue sections thoroughly in water Dehydrate the slides by i
4. probes upon their interac tion with the amyloid fibrils Thioflavin T has routinely been employed in this context However this dye is not an ideal predictor of the degree of fibrillization because its fluorescence varies substantially depending upon the structure and morphology of the amyloid fibrils Also the dye gener ates fairly high background and weak fluorescent signal in brain tissue sections Enzo Life Sciences ProteoStat Amyloid Plaque Detection Kit contains a novel 488 nm excitable red fluorescent molecular rotor dye to specifically detect amyloid plaques in cells and tissues The detection reagent inter acts with the cross sheet quaternary structure of amyloid fibrils and is readily excited by an argon ion laser source with emission maximum of 600 nm Relative to Thioflavin T this novel detection reagent demon strates significantly higher fluorescence emission intensity enhancement in the presence of amyloid protein fibrils and low non specific background It is also suitable for highlighting neurofibrillary plaques composed of f3 amyloid and tau proteins in brain tissue thin sections from animal mod els or from post mortem tissue sections of patients with Alzheimer s disease The ProteoStat Amyloid Plaque Detection Kit should facilitate the discovery of pharmaceuticals or therapies which prevent or slow Alzheimer s disease progression Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt
5. the kit should be stored at lt 20 C protected from light When stored properly these reagents are stable for one year from date of receipt Avoid repeated freezing and thawing Reagents provided in the kit are sufficient for approximately 40 tissue sec tions for analysis by fluorescence microscopy Reagent Quantity ProteoStat Amyloid Detection Reagent 10 uL Antifade Reagent 2mL 10X Assay Buffer 10 mL Additional Materials Required Tissue Sections For example post mortem brain tissue from patients with Alzheimer s disease from BioChain Institute Inc Hayward CA T1236039Alz for frozen tissue and T2236039Alz for paraffin embedded tissue and human adult normal brain tissue T1234041 for frozen tissue amp T2244040 for paraffin embedded tissue Standard fluorescence microscope Calibrated adjustable precision pipetters preferably with disposable plastic tips Glass cover slips Coplin jars or equivalent Xylenes or xylene substitute Thermo Scientific 9990507 for paraffin embedded tissue sections 50 70 90 and 100 Alcohol Reagent Grade 1 acetic acid in water Paraformaldehyde Deionized or distilled water Safety Warnings and Precautions This product is for research use only and is not intended for diagnostic purposes The ProteoStat Amyloid Detection Reagent contains DMSO which is read ily absorbed through the skin DMSO is harmful if ingested or absorbed through the skin and may cause ir
6. ation studies of amyloid plaques within paraffin embedded tissue sections of human brain tissue cerebellum from patients with Alzheimer s disease with fluorescein labeled antibodies as observed by fluorescence microscopy Panel A ProteoStat dye co localizes with Tau protein a Tau reactive antibody clone Tau 13 which recognizes human tau and specifically stains brain tissue early in Alz heimer s disease b ProteoStat Amyloid Detection dye stained tissue c composite image Panel B ProteoStat dye co localizes with beta amyloid a Beta amyloid reactive antibody clone 6E10 which specifically binds B amyloid aggregates b ProteoStat Amyloid Detection dye stained tissue c composite image VII References 1 Dedeoglu A Kubilus JK Jeitner TM Matson SA Bogdanov M Kowall NW Matson WR Cooper AJ Ratan RR Beal MF Hersch SM Fer rante RJ 2002 Therapeutic effects of cystamine in a murine model of Huntington s disease The Journal of Neuroscience 22 20 8942 8950 Ross CA Poirier MA 2004 Protein aggregation and neurodegenera tive disease Nat Med S 10 7 Gunilla T Westermark Kenneth H Johnson Per Westermark 1999 Staining methods for identification of amyloid in tissue Methods in Enzymology volume 309 pages 3 25 Brookmeyer R Gray S Kawas C 1998 Projections of Alzheimer s disease in the United States and the public health impact of delaying disease onset Am J Public Health 88 1337 42 Buc
7. ciantini M Giannoni E Chiti F Baroni F Formigli L Zurdo J Tad dei N Ramponi G Dobson CM Stefani M 2002 Inherent toxicity of aggregates implies a common mechanism for protein misfolding dis eases Nature 416 507 11 Biancalana M Koide S 2010 Molecular mechanism of Thioflavin T binding to amyloid fibrils Biochim Biophys Acta 1804 7 1405 1412 VIII Troubleshooting Guide Problem Potential Cause Suggestion Low ProteoStat dye staining in all treatments including positive control A low concentration of the ProteoStat Amyloid Detection Reagent was used or the reagent was incubated with the tissue for an insufficient length of time Either increase the re agent concentration or increase the incubation time Tissue sections were lost from the slide during speci men preparation and or staining procedures Avoid over vigorous wash ing with wash buffer from the squeeze bottle aim stream of buffer to the side of the tissue section and allow the buffer to flow gently over the tissue sec tions Non uniform or high background levels Loss of positive signal over time Excessive thickness of the tissue section improper washing or incomplete deparaffinization will cause high and non uniform back ground Water evaporated drying the coverslip onto the stained slide Ensure the optimum tissue thickness to be around 4 6 microns Use fresh xylenes for each batch of 5 slides and
8. claims must be made to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo CELLestial and ProteoStat are trademarks of Enzo Life Sciences Inc Several of Enzo s products and product applications are covered by US and foreign patents and patents pending Contents VII VIII Introduction en 1 Reagents Provided and Storage 1 Additional Materials Required 2 Safety Warnings and Precautions 2 Methods and Procedures 3 A REAGENT 3 B STANING TISSUE SECTIONS 3 Appendices eere eren 8 A FLUORESCENCE CHANNEL SELECTION FOR DATA 6 B EXPECTED enne 8 A liu 9 Troubleshooting Guide 10 Introduction Amyloid protein fibrils are characteristic of a variety of neurodegenerative diseases including Alzheimer s Huntington s and Parkinson s diseases as well as various transmissible spongiform encephalopathies It is diffi cult to predict the presence of amyloid deposits solely on the basis of a patient s clinical symptoms and thus histopathological analysis is often required post mortem to confirm a diagnosis This is often accomplished by measuring the optical properties of specific
9. deparaffinize only a few slides at one time Use the Antifade Reagent provided in the kit as a mounting medium Do not allow the coverslip to dry onto the stained slide Precipitate is observed in the 10X Assay Buffer Precipitate forms at low temperatures 10 Allow solution to warm to room temperature or 37 C then vortex to dissolve all precipitate Enzo Life Sciences www enzolifesciences com Enabling Discovery in Life Science NORTH SOUTH AMERICA ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler Pike Plymouth Meeting PA 19462 1202 USA T 1 800 942 0430 610 941 0430 F 610 941 9252 E info usaGenzolifesciences com SWITZERLAND amp REST OF EUROPE ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 441 061 926 89 89 F 441 061 926 89 79 E info chGenzolifesciences com www enzolifesciences com GERMANY ENZO LIFE SCIENCES GMBH Marie Curie Strasse 8 DE 79539 L rrach Germany T 49 0 7621 5500 526 Toll Free 0800 664 9518 F 449 0 7621 5500 527 E info deGenzolifesciences com www enzolifesciences com BENELUX ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium T 32 03 466 04 20 432 03 466 0429 E info be enzolifesciences com www enzolifesciences com incorporating NTERNA TONAL UK amp IRELAND ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK T 0845601 1488 UK cust
10. ncubating at room temperature in the following series of Coplin jars containing the following solutions for the times indicated Soak Number Reagent Duration of Soak 1 Deionized Water 1 minute 2 50 alcohol 1 minute 3 70 alcohol 1 minute 4 100 alcohol 1 minute Add one drop of Antifade Reagent warmed to room temperature then carefully apply a coverslip to the sample being careful not to introduce air bubbles Analyze the stained tissue sections by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard Texas Red filter set for imaging the amyloid plaques in post mortem brain tissue of patients with Alzheimer s disease VI APPENDICES A FLUORESCENCE CHANNEL SELECTION FOR DATA COLLECTION The selection of optimal filter sets for a fluorescence microscopy application requires matching the optical filter specifications to the spectral characteristics of the dyes employed in the analysis see Figure 1 Consult the microscope or filter set manufacturer for assis tance in selecting optimal filter sets for your microscope Predes igned filter sets for Texas Red should work well for this application o 0 c 5 9 o e 2 Figure 1 Fluorescence excitation and emission spectra for ProteoStat Amy loid Detection Reagent Spectra were determined in 1X Assay Buffer The absorption maximum of the dye is about 600 500nm while the emission maximum is Wavele
11. ngth nm approximately 600nm B EXPECTED RESULTS Alzheimer s disease a relatively common neurodegenerative disorder in the elderly is characterized by an accumulation of B amyloid AB and tau protein aggregates These two proteins can cause the loss of neurons and synapses Thioflavin T has routinely been employed for the determination of aggregated protein deposits both ex vivo and in vitro In the presence of amyloid fibrils ThT displays a strong increase in fluorescence intensity with excitation and emission maxima of roughly 440nm and 490nm respectively depending upon buffer composition The intercalation of ThT molecules into grooves between solvent exposed side chains within the characteristic crossed sheet structure of the amyloid fibril appears to lead to the increased fluorescence signal We found that the dye generates fairly high background and weak fluorescent signal in brain tissue sections as shown in Figure 2A The ProteoStat Amyloid Detection dye was developed to overcome the inherent limitations of commonly employed molecular probes for the assessment of protein aggregates in cells and tissues Like ThT the ProteoStat Amyloid Detection dye is a molecular rotor that binds 6 selectively to aggregated protein The dye s red fluorescence emis sion maximum of 600nm Figure 1 minimizes background problems associated with intrinsic or fixative induced auto fluorescence Be cause detecti
12. omers T 44 0 1392 825900 from overseas F 44 0 1392 825910 E info uk enzolifesciences com www enzolifesciences com www enzolifesciences com FRANCE ENZO LIFE SCIENCES c o Covalab s a s 13 Avenue Albert Einstein FR 69100 Villeurbanne France T 33 472 440 655 F 33 437 484 239 E info fr enzolifesciences com www enzolifesciences com Alexis assay designs Stressgen
13. on of amyloid plaques with the ProteoStat dye is based upon obtaining fluorescent signal at longer wavelengths than used for ThT dye it enables the quantification of amyloid fibrils in the pres ence of colored and fluorescent compounds that interfere with fluo rescence signal generated by ThT Figure 2B highlights the substan tial reduction in background noise afforded by the ProteoStat dye relative to ThT A Thioflavin T Normal brain tissue Brain with Alzheimer s disease B ProteoStat dye Normal brain tissue Brain with Alzheimer s disease Figure 2 Detection of amyloid plaques within paraffin embedded tissue sections of human brain tissue cerebellum from patients with Alzheimer s disease and human adult normal brain tissue cerebellum with 1 uM Thioflavin T dye panel A and ProteoStat Amyloid Detection dye panel B as observed by fluorescence microscopy In addition the use of antibodies directed against B amyloid or tau protein in conjunction with the ProteoStat dye confirm the selectiv ity of the probe for detection of amyloid plaques in post mortem brain tissue of patients with Alzheimer s disease Figures 3A and 3B page 8 A ProteoStat dye co localizes with Tau 13 protein a Tau 13 Antibody b ProteoStat dye composite image B ProteoStat dye co localizes with Beta Amyloid a Antibody b ProteoStat dye c composite image Figure 3 Co localiz
14. ritation to the eyes Observe appropriate precautions when handling these reagents Reagents should be treated as possible mutagens and should be handled with care and disposed of properly Observe good laboratory practices Gloves lab coat protective eyewear should always be worn Never pipet by mouth Do not eat drink or smoke in the laboratory areas All blood components and biological materi als should be treated as potentially hazardous and handled as such They should be disposed of in accordance with established safety procedures To avoid photobleaching perform all manipulations in low light environ ments or protected from light by other means V Methods and Procedures NOTE Allow all reagents to thaw at room temperature before starting with the procedures Upon thawing gently hand mix or vortex the reagents prior to use to ensure a homogenous solution Briefly centrifuge the vials at the time of first use as well as for all subsequent uses to gather the contents at the bottom of the tube A REAGENT PREPARATION 1 1X Assay Buffer Allow the 10X Assay Buffer to warm to room temperature Make sure that the reagent is free of any crystallization before dilution Prepare enough 1X Assay Buffer for the number of samples to be assayed by diluting each milliliter mL of the 10X Assay Buffer with 9 mL of deionized water 4 Formaldehyde Solution The following procedure is for preparation of 10 mL of 4 formal
Download Pdf Manuals
Related Search