Hi, I've realized a IHC for NGF, and works well, maybe my protocol works for you too.
(criostate slides of 40 micrometers)
Wash 15 min in PBS (0.1M, pH 7.4) at RT
Incubate 15 min in 0.3% H2O2 (in PBS) at RT
Wash 5 min in PBS at RT
Incubate 3 times in EtOH 50% (in PBS), 10 min each, at RT
Wash 15 min in PBS, at RT
Incubate 1 hr in PBS + 0.2% Triton x-100 / 5% Normal Serum at RT
Incubate with primary antibody (1:500), in PBS + 0.2% Triton x-100 / 2% Normal Serum at 4 C O.N.
Wash 3 times in PBS , 15 min each, at RT
Incubate with secondary biotinylated antibody (1:500), in PBS + 0.2% Triton x-100 / 0.1% NS, 2 hrs at RT
Wash 3 times in PBS , 15 min each, at RT
Incubate one and a half hour with ABC Kit (1:110) diluted in PBS, at RT (mix 30 min before use)
Wash 3 times in PBS , 15 min each, at RT
Incubate in 0.04% DAB + 0.01% H2O2 + NiCl2 0.06%, 10 min at RT (discard to chlorine solution)
Wash 3 times in PBS , 15 min each, at RT (discard to chlorine solution)
