[Histonet] IHC on 3 year fixed tissue?

Jacqui Detmar detmar <@t> lunenfeld.ca
Sat Mar 13 21:18:17 CST 2010

Hey there.  I have done IHC on mouse placentae that have been fixed in NBF for over 2 years and it took me 5-10 minutes of ProK (10 ug/ml in PBS) followed by 60 minutes of HIER in 10 mM citrate buffer (made sure PBS was at pH 6) in a veggie steamer.  
I had previously done this IHC in a veggie steamer for only 25 minutes in 10 mM citrate buffer (pH 6) for mouse placentae that had only been fixed for 24 hours in NBF.  The antigen was a nuclear receptor (AhR); I have found nuclear antigens to be slightly more difficult to retrieve...at least for mouse placenta!  Note that the 2-year-fixed tissue did NOT react positively until the 60-minute mark after HIER, although I tested at 25, 35 and 50 minutes.    Only 60 minutes HIER plus enzyme retrieval worked, even though "quick-fixed" tissue worked simply with 25 minutes HIER.  Definitely worth a try, if you're desperate.  It took me over a year to get this **#&%*#&%** tissue working! <grin>   Also, used a  1:100 dilution instead of my normal 1:500 dilution of the antibody, so you might want to titrate the antibody, too. 
Am I being lucid?  Have just come home from a most excellent dinner party!  Perhaps not such a good idea to give histo-advice on a Saturday night....but I deeply sympathized with your plight <grin>.  If you have any questions, please feel free to contact me personally, Kevin, either on gmail or at my SLRI account.
Jacqui Detmar
Work phone:  1-416-646-0223
Work fax:      1-416-862-9696
Cell phone:    1-647-273-8735
jacquidetmar <@t> gmail.com


From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Rene J Buesa
Sent: Sat 3/13/2010 6:17 PM
To: histonet <@t> lists.utsouthwestern.edu; Kevin Egan
Subject: Re: [Histonet] IHC on 3 year fixed tissue?

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.


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