Immunohistochemistry c-fos

Reagents

This protocol has been tested on formalin-fixed (10%) free floating brain sections (25 um-thick).

DAY 1 All steps done at RT on orbital shaker.

1. Rinse the sections in 1 X PBS for 3 times, 10 min each.

2. Wash in 0.3% H2O2 for 25 min. This steps blocks endogenous peroxidase activity.

3. Rinse in 1 X PBS for 3 times, 10 min each.

5. Incubate sections with primary antibody at RT overnight. Primary rabbit antibody against c-fos is diluted 1 : 50,000 in 1 X PBT with 3% normal donkey serum.

DAY 2

6. Rinse with 1 X PBS for 3 times, 10 min each.

7. Incubate sections with biotinylated-conjugated donkey anti-rabbit antibody of 1 : 1,000 in 1 X PBS.

Note: Make ABC solution (10 ul A + 10 ul B in 5 ml 1 X PBS) and allow to sit at RT for at least 30 min before use.

8. Rinse with 1 X PBS for 3 times, 10 min each.

9. Incubate in ABC solution for 1 hr.

10. Rinse with 1 X PBS for 2 times, 10 min each.

11. Incubate in 0.04% DAB and 0.01% H2O2 for about 4 min. Check every minute. Depending on the antibody and antigen levels, time for DAB development will vary (500 ul DAB + 50 ml PBS; right before use add 16 ul of 30% H2O2). For dark-blue color: add 100 ul 5% Cobalt and 500 ul 1% Nickel (method of Shu and colleagues, 1988).

12. Rinse with 1 X PBS for 2 times, 10 min each.

13. Store sections in PBS-Azide or mount onto gelatin coated slides.

Contributed by L. Gautron.


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