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Immunofluorescence on parrafin-sections of colon and colorectal cancer


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#1 SteveW

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Posted 17 March 2011 - 07:12 AM

Hi
Can anyone give me some advice on immunofluorescence on formalin-fixed parrafin-embedded sections of human colon and colorectal cancer please.
I'm clearing in Clearene, graduated alcohols then distilled water.
Antigen retrieval with EDTA buffer heated for 15mins (have also tried citrate buffer and pepsin digestion)
Blocking with casein (and have also tried secondary host serum)
Incubating with primary antibodies to CD3/CD4/CD8 and FoxP3 for 1hr room temp which I KNOW WORK ON SAME SECTIONS using immunohistochemistry.
Then adding secondaries - using goat anti-rabbit or anti-mouse F(Ab)2 fragment antibodies conjugated to FITC or TxRed and various dilutions.
I'm getting a fair amount of background (although I don't think a large amount?!) but just not seeing any staining!!!

I can't understand how it works with the histochemistry but not with the fluorescence! Is there anything specific to bowel that I need to do?
Any tips or tricks would be appreciated.
Thanks very much

Steve

#2 bob1

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Posted 17 March 2011 - 01:05 PM

The formaldehyde may be causing some autofluorescence, try washing in 0.1 M glycine in PBS for 24 hours with several changes of the glycine.




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