We use acetone/ethanol (as recommended by Gayle Callis in one of her NSH classes). The slides look great; nice crisp nuclei (which you just won't get with acetone alone). We air dry the slides and fix for 10 minutes in RT acetone/ethanol (75% acetone/25% absolute ethanol) and then put the slides into wash buffer and continue with IHC.
I have heard that this fixative could be problematic with some antigens, but we have not found that to be the case with anything we have used in my lab. Insulin should be fine. Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ________________________________ From: Patti Loykasek <[EMAIL PROTECTED]> To: "FU,DONGTAO" <[EMAIL PROTECTED]>; histonet@lists.utsouthwestern.edu Sent: Wednesday, December 3, 2008 11:38:07 AM Subject: Re: [Histonet] IHC on fresh frozen After hearing a presentation by Sharon Lear describing some low temp antigen retrieval that she did, we changed our method for frozen sections. We fix the frozen sections in 10% nuetral buffered formalin for 30'-60', rinse, then do a gentle pretreatment. The gentle pretreatment is usually 10mM citrate buffer pH6 at 70 degrees C for 30'. Slides are cooled, and usual IHC done. This has worked well for us. Our staining with this method is more reliable & intense than with previous methods. Patti Loykasek > Hi, all > > Recently I did some IHC(chromagen methods) on mouse fresh frozen > tissues, mainly using insulin antibody on pancreas. The image is > much fuzzier compare to paraffin embedding tissue. And the > staining also smeared to acinar cells which surround the islet. > > I airdried slide(>30min) and used a general acetone method(-20C > 5min) to fix the tissue before I did IHC. > > How can I get a relatively sharp staining on the fresh frozen > tissue?Does anyone here have any experience on it? Any > suggestions? > > Many thanks and have a nice day, > > Ann > > > > > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > This e-mail message, including any attachments, is for the sole use of the intended recipients and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by e-mail and destroy all copies of the original message, or you may call PhenoPath Laboratories, Seattle, WA U.S.A. at (206) 374-9000. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet