Why is TUNEL staining not working with my antibodies (IHC)?

02 December 2016 0 3K Report

Hi all, I have tried to do TUNEL staining from the Sigma/Roche kit and it works beautifully and it appears to be compatible with some antibodies but not others. I know for sure that my antibodies are good.

The Roche protocol consists of...

PBS wash

Permeabilization with 0.1% Triton X-100 in 0.1% sodium citrate on ice for 2 mins

Boil with 0.1M Sodium citrate (pH 6.0)

PBS Wash

0.1M Tris-HCl (pH 7.5), containing 3% BSA and 20% normal bovine serum for 30 minutes at RT

TUNEL label for 1 hour at 37C

PBS Wash

And then I follow the normal IHC protocol

Block with 3% Normal Donkey Serum and 0.2% Triton-X

Primary antibody

Wash

Secondary Antibody

Wash

What is the problem? I've tried decreasing the sodium citrate level to 10mM instead, which is what I normally use and it still doesn't work.

The only step I'm worried about is "0.1M Tris-HCl (pH 7.5), containing 3% BSA and 20% normal bovine serum", do you guys think that might have any impact on antigen recognition?

Thanks!

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