[Histonet] IHC on 3 year fixed tissue?

[Rene J Buesa]

I have to imagine that you are doing the HIER in the FFPE sections? If you have used autoclave unsuccessfully it seems that the epitopes are so cross-linked that they do not respond to the treatment.
I would try a piece of fixed tissue before processing, cut it in a very thin slice (about 2 mm maximum) and heat it in distilled water at 65ºC for 30 minutes. Wah it thoroughly and process it as usual.
After that, prepare the sections and HIER them in citrate buffer in a steamer and proceed as usual.
By doing this you will have to steps were the cross-linkages area going to be weaken and perhaps you will get some acceptable reaction.
René J.

--- On Sat, 3/13/10, Kevin Egan <kevinpe <@t> mail.med.upenn.edu> wrote:


From: Kevin Egan <kevinpe <@t> mail.med.upenn.edu>
Subject: [Histonet] IHC on 3 year fixed tissue?
To: histonet <@t> lists.utsouthwestern.edu
Date: Saturday, March 13, 2010, 2:15 PM


Hello Histonetters,

I've been asked to perform anti-GFP IHC on some mouse bones which have been sitting in NBF for 3-4 years. My Abcam rabbit polyclonal gets great results in GFP mouse control tissue, but I can't seem to get any results out of these bones. I've tried citrate buffer HIER using autoclave, hotplate, and 65 degree water batch over night, but my signal is non-existent (or really the noise is so huge you can't distinguish).Do the great minds of histonet have any ideas on how to achieve good staining? Is enzymatic epitope retrieval worth  a try? Does anyone have a time machine I can use to stop my predecessors from just sticking the bones in NBF and forgetting about them for years?

This listserv has helped me countless times, I am truly appreciative of the knowledge and experience available on Histonet.

Thanks,
Kevin

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