Immunohistochemistry c-fos
Reagents
- Rabbit anti-c-Fos antibody from Oncogene/Calbiochem #PC38.
- 0.3% H2O2 solution: 100 ul 30% H2O2 in 10 ml 1 X PBS.
- Biotinylated-conjugated donkey anti-rabbit antibody from Jackson Immunoresearch #711-065-152.
- Vectastain Elite ABC Kit from Vector Laboratories #PK-6200.
- 1 X PBS, 27.6 g Sodium Phosphate, dibasic, 160 g Sodium Chloride, 20 L ddH2O, pH to 7.4 with HCl.
- PBS-Azide, 0.8 g Sodium Azide in 4 L 1 X PBS.
- DAB solution: diaminobenzidine tetrahydrochloride from aldrich-sigma (comes in powder, dissolve in PBS, store aliquots of a 4% stock solution at -20c). Nickel and cobalt: stock solution of 1% nickel ammonium sulfate (Fisher Scientific #N48-500) and 5% cobalt chloride (Fisher Scientific #19403580) are prepared in 1 X PBS in advance and stored at RT.
- Normal donkey serum from Jackson ImmunoResearch #017-000-001.
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.