1.  Important: Do not allow sections to dry between the following steps!!!

2.  Put sections into wells containing  1 ml of PBST (phosphate buffered saline, 0.9% NaCl, 0.3% Trition X-100, pH7.4).  (24 well tissue culture plate is a convenient dish).

3.  Wash sections for 10 min with slight agitation on a rocking platform.

4.  Replace PBST with 1 ml of fresh one and continue wash for another 10 min.  (This step is important to remove sodium azide and to increase tissue permeability).

5.  To abolish endogenous peroxidase activity, incubate sections in 1 ml of cold (4°C) 100% methanol containing 1% of hydrogen peroxide for 30 min on a rocker.  (Make up always fresh!).

6.  Wash sections in PBST 2 times for 5 min each.

7.  Incubate sections in 0.5 ml of Blocking solution (PBST + 10% normal goat serum) for 1 hour.

8.  Incubate sections in primary antibodies diluted in Diluting solution (PBST + 10% normal goat serum) overnight on a rocker at room temperature.  (Can do this step over weekend in a cold room).

9.  Next day, first, prepare secondary antibody solution and ABC reagent, then proceed with the following steps.

10. Remove primary antibody solution.

11. Wash sections 3 times in 1 ml of PBST for 20 min each on a rocker.

12. Incubate sections in secondary antibodies solution for 1 hour (can do this step overnight in a cold room). 

13. Remove secondary antibodies.

14. Wash sections 3 times in 1 ml of PBST for 20 min each on a rocker.

15. Incubate sections in ABC reagent for 2 hours (can do this step overnight in a cold room, but background may be a problem).

16. Remove ABC reagent. 

17. Wash sections 3 times in 1 ml of PBST for 20 min each on a rocker.

18. Prepare DAB substrate solution.  Do not add hydrogen peroxide!!!

19. Remove completely PBST.  Add hydrogen peroxide to DAB substrate solution mix by inverting 2 times and add 0.5 ml to sections.

20. Watch the reaction.  Color normally develops within first minutes.  You can monitor it under microscope.

21. Remove substrate solution.

22. Stop the reaction by washing sections in distilled water 3 times for 2 min each (if background is a problem you can wash them in water from several hours to overnight).

23. Mount sections on glass slides, air dry overnight.

24. Next day incubate in Histoclear (make sure you use fresh one) 2 times for 10 min each

25. Coverslip slides with Pro-Texx (Baxter) and let them dry.

NOTES

1.   Secondary antibody solution.  Dilution:  1:400

Add 1 drop (50 ml) of secondary biotinylated antibody stock solution (blue bottle from ABC kit) to 20 ml of Diluting solution (PBST + 10% normal goat serum).

2.   ABC reagent. 

Add 2 drops (100ml) of reagent A from ABC Elite kit to 10 ml of PBST and mix by inverting 2 times, then add 2 drops of reagent B and repeat inverting 2 times.  Let this solution sit for at least 1 hour.

3.     DAB substrate solution. 

1)     Add 2 drops of Buffer Stock Solution (from DAB substrate ABC kit) to 5 ml of distilled water and mix well by inverting.

2)     Add 4 drops of DAB Stock Solution and mix well by inverting.

3)     If you want a black stain, add 2 drops of the Nickel Stock Solution and mix well.

4)     Just before applying to sections add 2 drops of the Hydrogen Perioxideee Solution and mix well.

5)     Apply 0.5 ml of the Substrate Solution to tissue sections.

Controls

Always include three controls:

1.     Sections processed without primary antibody step.

2.     Sections processed without primary and secondary antibody steps.

3.     Sections processed without  primary,  secondary, and ABC reagent steps.

Optional controls: (a) preincubate primary antibody with your target protein;

(b) use non-immune serum instead of primary antibody (dilution the same as primary antibody).

Troubleshooting suggestions

If staining is weak or abcent:

1.     Reduce Triton X-100 concentration to 0.1%, or use for Diluting solution PBS instead of PBST.

2.     Make sure pH of your PBST or PBS is not lower than 7.4 (primary antibody will not bind well in acidic pH)

3.     Increase concentration of primary antibody.

If background staining is a problem:

1.     Make sure concentration of NaCl in PBST or PBS is at least 0.9% (=0.15M); to prevent nonspecific binding of ABC reagent increase NaCl to 0.3-0.5M.

2.     Perform Avidin/Biotin blocking step (Vector protocol).

3.     Try different dilution of primary antibodies.

Alexander Murashov

GOOD LUCK!