Autoclave method
a. Deparaffinize the section and rehydrate in PBS
b. Immerse the slides in a autoclavable tray, containing 100 mM Tris-HCL pH 10.
c. Autoclave at 120 C for 10 minutes.
d. Let the tray cool down at room temperature.
e. Wash the slides twice in PBS for 2 minutes
3. Block endogenous peroxidases:
a. Soak slides in 3% H2O2/PBS for 10-15 minutes at room temp.
b. Wash 3 x 5’ with 1X PBS.
4. Shake and wipe off excess 1X PBS. Circle all sections with a PAP pen. Add 50-75 ul of
blocking buffer to each section immediately, so that the sections don’t dry out. Don’t touch
sections with tip.
Blocking buffers: 5% BSA/0.5% Tween-20 in 1X PBS
3% BSA in 1X PBS
3% BSA/0.1% Tween in 1X PBS
MOM (for mouse and rat monoclonal antibodies, use Molecular
Probes secondary antibodies with MOM basic kit)
Note: A lot of researchers would like to use 2.5-10% Normal Serum/0.5% Tween-20 in 1X PBS as a blocking buffer.
5. Incubate 1 hour at room temperature or overnight at 4ºC in a humidified chamber. Do not let the
slides touch each other.
6. Dilute primary antibody in blocking buffer (dilutions vary depending on your antibody and expression of the antigen).
Discard the blocking buffer, but don’t wash. Add 50-75 ul per section and incubate 1 hour at room temperature or overnight at 4ºC in a
humidified chamber.
7. Drain primary antibody off section. Wash slides 3 x 5´ in 1X PBS. (You may need to
wash slides in 1X PBS + 0.1%-0.5% Tween-20 for some primary antibodies)
8. Dilute biotinylated secondary antibody 1:150 to 1:750 in blocking buffer. Add 50-75 ul per
section and incubate 45´-1 hour at room temperature in a humid chamber.
9. Drain secondary antibody and wash slides 4 x 5’ in 1X PBS.
For using secondary antibodies that are peroxidase conjugated, skip to step 11.
10. Add 1 drop of ABC (Ready to Use; Vector to each section and incubate samples for 30
minutes at room temperature. Wash 3 x 5 minutes in 1X PBS
11. Make DAB according to Vector protocol in ddH2O. WEAR GLOVES.
2.5 ml dd H2O
1 drop buffer; mix
2 drops DAB; mix
1 drop H2O, mix.
(If you want a gray-black stain, add 2 drops of the Nickel Solution; mix)
Add immediately to slides and wait for color change (approximately 2-10 minutes).
Drain slides and wash with dd H2O for 5 minutes.
Dispose of DAB waste with bleach.