- Deparaffinize slides in two changes of xylene for five minutes each.
- Wash slides in 100% alcohol twice for two minutes each.
- Wash slides in 70% ETOH for five minutes.
- Wash with PBS (1% horse serum) two times x five minutes.
- 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
- 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.
- Cool down for 20 minutes at RT
- Wash with PBS with 1% horse serum two times x five minutes.
- Circumscribe tissues with a vaseline pen to contain antibodies.
- 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).
- Wash two times with PBS 1% horse serum three to five minutes.
- Primary Antibody working solution incubates one to two hours at room temperature or at 4° overnight.
- Wash two times with PBS with 1% horse serum three to five minutes.
- Secondary antibody 60 minutes at room temperature.
- Wash two times with PBS with 1% house serum thre to five minutes.
- Third antibody 60 minutes at room temperature.
- Add DAB from Dako x five to ten minutes using bulb pipette (timing is crucial).
- Wash two times with PBS three to five minutes.
- Apply Hematoxylin counterstain for 30 seconds (immerse in solution).
- Wash for five minutes in running water.
a. 95% ETOH dip a few times x1
b. 100% ETOH x one minute times two
c. Xylene 100% two-five minutes xtwo
- Coverslip with mounting media (per mount).