[Histonet] Problems with IHC/DAB on tumours

Martin S. sonya.martin <@t> soton.ac.uk
Wed Mar 14 07:05:42 CDT 2007

Hi All,
I'm having a problem doing IHC/DAB on frozen colorectal tumour samples.
I'm getting a lot of positive cells in sections even without without any
antibodies or ABC. I've narrowed it down to the DAB but I dont think its
DAB ppt as its not random - you can see the cells rapidly stain under
the microscope after adding the DAB - it only takes a matter of seconds!
I've been using DAB from Sigma and also Vector - both show the same
result (I have filtered both with 0.2um filter). I do not see it in
mouse liver or spleen tissue (frozen in OCT in a bath of isopentane on
dry ice - the CR tumour is human and was snap frozen in liquid
nitrogen). I dont think its endog peroxidase as it occurs even if you
add the DAB diluted in peroxidase suppressor (Pierce - has always worked
well for other tissues).
Any suggestions? 
Is there something else that could be in the tissue that the DAB would
react with? 
Would using AEC instead help?
The Pierce suppressor is supposedly better than H2O2 methods - can
anyone recommend other peroxidase inhibition protocols?

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