I tried CD11b antibody immunohistochemistry on frozen-section 4% PFA fixed tissues (post-fixed for 48hrs) using the following protocol:
1% H2O2 in PBS and washed.
Preincubution in 5% Normal Horse serum in Triton-X.
Primary antibody (Dilution: 1:500 using Serotec: mouse anti rat CD11b Antibody overnight at 4 °C. Washed
Secondary antibody (Dilution: 1:200 using Vector: Horse anti mouse IgG. Washed.
ABC and washed.
DAB and washed.
Dehydrated, cleared and mounted in entallan
Results:
Staining Pattern: scanty and faint appearance of microglia around hypothalamus, hippocampus, but not in the neocortex.
I tried antigen retrieval (citrate buffer (pH 6.0) in microwave), result was worse.
What did I miss?
I work with avian LB fixed for 48 hs, I dont´t know if my protocol serves to you. But I can say that the blood is processed, puriffy and stained with the AV20-fit C and then I fixed with PFA.
I hope you serve the answer.
Marìa Fernanda Peralta
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