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| General Staining Procedure for IHC |
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General Staining Procedure for IHC on FFPE Tissue Section Using Acu-Stain HRP kits 1. Bake slides in 55 – 60°C oven for 30 minutes to overnight 2. Deparaffinize slides in two baths of xylene, 5 minutes each 3. Rehydrate in two baths of 100% ethanol, one bath of 95% and 80% ethanol, 3 minutes each 4. Eliminate endogenous peroxidase in 3% H2O2 in water, 10 minutes 5. Rinse slides in 1X PBS or water 6. Perform appropriate pretreatment (HIER or enzyme digestion) 7. Rinse slides 3 times with 1X PBS, 2 minutes each 8. Apply blocking solution, incubate 10 minutes (DO NOT RINSE OFF) 9. Apply 1° antibody, incubate 30 – 60 minutes 10. Rinse slides 3 times with PBS-T, 2 minutes each 11. Apply 2° antibody, incubate 10 minutes 12. Rinse slides 3 times with PBS-T, 2 minutes each 13. Apply enzyme conjugated streptavidin, incubate 10 minutes 14. Rinse slides 3 times with PBS-T, 2 minutes each 15. Apply substrate-chromogen solution, incubation time varies with each reaction (e.g. 5 minutes for DAB, 10 minutes for AEC, etc.) 16. Rinse slides under running tap water, 2 minutes 17. Counterstain slides in Meyer’s Hematoxylin, 2 minutes 18. Rinse off excess Hematoxylin under running tap water 19. Dip slides in 1X PBS 10 times 20. Rinse slides under running tap water, 30 – 60 seconds 21. Dehydrate slides in one bath of 80% and 95% ethanol, then two baths of 100% ethanol, 10 dips each 22. Dip slides in two baths of xylene, 10 dips each 23. Coverslip slides 24. Microscopy
All steps done in room temperature, unless otherwise specified.
FFPE: formalin-fixed, paraffin-embedded HIER: Heat Induced Epitope Retrieval PBS-T: 1X PBS with 0.01% Tween 20
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