Author Topic: Problem with IHC for MHCII  (Read 3719 times)

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Offline Rubendario

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Problem with IHC for MHCII
« on: April 06, 2012, 02:26:33 PM »
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


« Last Edit: April 06, 2012, 04:40:59 PM by Rubendario »

Problem with IHC for MHCII
« on: April 06, 2012, 02:26:33 PM »

Offline Herpes

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    • http://www.bioworldantibodies.com
Re: Problem with IHC for MHCII
« Reply #1 on: April 08, 2012, 10:13:07 AM »
We use the anti-MHC II antibody from Abcam with a one step protocol for deparaffinization and antigen retrival. It works for us very well.
The protocol can be downloaded from http://www.bioworldantibodies.com/info/IHC.pdf

Re: Problem with IHC for MHCII
« Reply #1 on: April 08, 2012, 10:13:07 AM »