Hello, On Thu, Feb 21, 2013 at 8:00 PM, <histonet-requ...@lists.utsouthwestern.edu> wrote: > From: "Margaryan, Naira" <nmargar...@luriechildrens.org>
> I am trying to do IHC on FFPE 4 weeks old zebrafish with different Abs. Is > there a trick > working with zebrafish? I am using the same IHC protocol I always use on > human and > mouse tissue and my Abs are suppose to work on fish as well as human. I run > human > and fish sections together with same AR and IHC protocol. By the end I get > beautiful > staining on human section and or nothing or some > fuzzy/fuggy/unclarified/undistinguished reaction. I suggest you prepare some unfixed frozen sections for comparison. If the antibody reacts to the unfixed tissue but not the fixed then you probably need to perform one or another antigen retrieval method. I have been having good success with http://www.ncbi.nlm.nih.gov/pubmed/21603650 For my fish, Nothobranchius, I am now fixing for two days at 4 oC (in PFA) and then decalcifying 15% EDTA (pH 7.3) for three days (the last day in 30% sucrose dissolved into the 15% EDTA) before freezing in liquid nitrogen for sectioning. This method is maintaining the structure beautifully. As regards using the above antigen retrieval method I have both soaked the tissue (after fixing) in the 70 oC pH 9 TRIS and then decalcified as well as decalcified, cryo-protected and sectioned (APTES coated slides) and then performed the antigen retrieval at 70 oC. Both routes work well for my fish and the sections don't detach. If you cannot carefully dissect out the tissue of interest then I suggest decalcifying the tissue. You only need one piece of bone to catch on the blade and rip through the tissue to cause a lot of frustration and waste a specimen. I hope this helps. -- Tyrone Genade Ph.D. Department of Human Biology University of Cape Town South Africa http://tgenade.freeshell.org email: tgen...@gmail.com tel: +27-84-632-1925 (c) ******************************************************************************** Romans 6:23: The gift of God is eternal life through Christ Jesus our Lord. To find out how to receive this FREE gift visit http://www.alpha.org. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
