I have problems with the inmunochemistry for MHCII in brain tissue, I designed the next protocol, and I donīt have mark in my positive control, the same protocol was effective for GFAP (1:50, DAKO monoclonal raised in mouse)
dewaxing 15 min Xilol
serial graded alcohol 100% 3 min, 96% 3 min, 70% 3 min, 50% 3 min, 30% 3 min, PBS 5 min
Antigen retrieval 30 min bath water in Tris-EDTA pH 9 at 95°C
Quenching Peroxidase 10 min in 3% H2O2 in water
Permeabilization 1 h PBS 1X Triton-X100 0.3% BSA 1% Normal Serum 10%
primary antibody 1:50 MHCII anti-rat MCA2687 monoclonal raised in mouse in PBS 1X overnight 4°C
secondary antibody 1:200 anti-mouse biotinylated 1 h
Enhancing reaction 1 h ABC kit Elite, and revealed with DAB kit Vector 3-5 min
I changed the buffer in antigen retrieval step for citrate pH6 following recomendations of Serotec (
http://web.uk-plc.net/abd/product/ox-6-anti-mhc-class-ii-h-2i-ak-s-antibody-mca2687ga.html), but I obtained the same result, the same antibody was effective in tissues obtained by vibratome, in this time my tissues was paraffin-embedded
