Immunohistochemistry Protocol

1. Deparaffinize slides in two changes of xylene for five minutes each.
2. Wash slides in 100% alcohol twice for two minutes each.
3. Wash slides in 70% ETOH for five minutes. 
4. Wash with PBS (1% horse serum) two times x five minutes.
5. Block endogenous peroxidase activity by incubating for 10 minutes in 3% H2O2. Rinse two-three times in water.
   a.  Not required if you use Dako dual endogenous Enzyme Block reagent
6. Place slides in a plastic coplin jar or staining dish with the BD Retrievagen A working solution and heat to 193 F in a microwave 
   a.  Heat for five minutes at a power setting of seven.
   b.  Replace evaporated water.
   c.  Heat for five minutes at a power setting of seven.
7. Cool down for 20 minutes at RT
8. Wash with PBS with 1% horse serum two times x five minutes.
9. Circumscribe tissues with a vaseline pen to contain antibodies. 
10. Add blocking Reagent to slides for ten minutes at RT using a bulb pipette to fill vaseline circle.
    a. Dual Endogenous Enzyme Block (includes H2O2) or 3% H2O2.
    
    If background is unacceptable, we can use an additional serum free blocking step (Dako serum-free protein block).
    
11. Wash two times with PBS 1% horse serum three to five minutes.
12. Primary Antibody working solution incubates one to two hours at room temperature or at 4° overnight.
13. Wash two times with PBS with 1% horse serum three to five minutes.
14. Secondary antibody 60 minutes at room temperature.
15. Wash two times with PBS with 1% house serum thre to five minutes.
16. Third antibody 60 minutes at room temperature.
17. Add DAB from Dako x five to ten minutes using bulb pipette (timing is crucial).
18. Wash two times with PBS three to five minutes.
19. Apply Hematoxylin counterstain for 30 seconds (immerse in solution).
20. Wash for five minutes in running water.
21. Dehydrate
    a.  95% ETOH dip a few times x1
    b.  100% ETOH x one minute times two
    c.  Xylene 100% two-five minutes xtwo
22. Coverslip with mounting media (per mount).