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Author Topic: Transcription factor in lymphoid tissue sections  (Read 3900 times)

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Offline aino

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Transcription factor in lymphoid tissue sections
« on: January 21, 2005, 05:11:39 AM »
Hello,

I am a PhD student and new in the immunohistochemistry area. My problem is the non-detection of a Kruppel-like zinc finger transcription factor (similar to Bcl6) in frozen sections of spleen, lymph nodes and lung. I have a polyclonal rabbit antibody that works on 4% PFA-fixed cytospins on fibroblasts transduced with the protein, and also works well on westerns (I know this may be irrelevant to sections). However, I can not detect it on either PFA- or acetone fixed frozen sections - not even with a combo of an primary Ab + secondary biotinylated Ab + anti-biotin sheep Ab + biotinylated donkey anti-sheep Ab + ABC-alkaline phosphatase. All Abs except the primary are known to work well at the concentrations used.

Now, could it be that the ag is masked, for example by DNA, and I would be better of eg. doing formalin-fixed paraffinised sections followed by antigen retrieval? Or should I try just incubating the Ab for much longer? Or try treating frozen sections with acid to denature DNA as some do for BrdU detection? Also, although the Ab works on cytospins, I've been told that cytospin staining may not reflect staining on frozen sections fixed in a same way - why would this be?

It would be great to get an opinion from someone who has experience on staining for a (likely low-copy) transcription factor.

BIG THANKS!

Aino

Transcription factor in lymphoid tissue sections
« on: January 21, 2005, 05:11:39 AM »