Tissue is fixed in PFA, sliced at 30 microns on a cryostat and free floated. Blocking agent is 1% BSA, 0.3% Triton X, 2% Donkey Serum. Incubated in 1Ab 24 hours, 2Ab 2 - 24 hours. Using maximum recommended concentration. I have tried it without the antigen retrieval, this is my next step and it has not been that successful with the one sanitgen retrieval method I have tried (used 2% Nonfat milk, 0.3% Triton X, 0.1% Sodium Azide in PBS, off of ImmunoWorld web site).