1. Deparaffinize slides in 2 changes of xylene, one for 40 minutes, the other for 20 minutes.
2. Transfer slides to 100% alcohol, 95% alcohol, 80% alcohol, 60% alcohol and ddH2O for 5
minutes each.
3. Place slides in vessel filled with antigen retrieval buffer and microwave on middle for several
minutes (700 W oven), allow retrieval solution to cool at room temperature.
4. Rinse 2 x 5 min with TBS.
5. Block endogenous peroxidase activity by incubating sections in 3% H2O2 solution in methanol
for 30 minutes.
6. Rinse 2 x 5 min with TBS.
7. Block in 5-10% goat serum in TBS for 30 minutes at room temperature.
8. Drain slides for a few seconds (do NOT rinse) and wipe round sections.
9. Apply primary antibody made up in TBS. Incubate 1 hour at room temperature or 4°C
overnight.
10. Rinse 2 x 5 min with TBST.
11.Apply secondary biotinylated antibody made up in TBS for 1 hour at room temperature.
12. Rinse 2 x 5 min with TBS.
13. Apply streptavidin peroxidase for 15 minutes or 30 minutes at room temperature.
14. Rinse 2 x 5 min with TBS.
15. Develop with chromogen (DAB) at room temperature, watching under microscope.
16. Rinse in running tap water for 5 minutes.
17. Counterstain in mayor’s hematoxylin bath for 30-60 seconds.
18. Wash in water bath 7-8 times, then tap water for 3 minutes.
19. Dehydrate through 60%, 80%, 95% and 100% alcohol for 5 minutes each. 20. Transfer to
xylene for 5 minutes. Air for 30 minutes.
21. Mounting.
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