Robin Have you tried other retrieval methods, its best to normally try to stay away from HIER on bone but sometimes you have to use it. If you have not already tried other retrieval methods I would try no retrieval, proteinase K and then trypsin or pronase, my favorite is proteinase K. Since the samples have been decaled we have sometimes noticed in our hands that the nuclei can get "chewed up" a bit with enzyme retrieval so if you notice that you may need to back off on the time of retrieval, just as long as you do not loose staining intensity. If this antibody will not work with any other retrieval method then you will have to use HIER retrieval. Instead of retrieving at 95 or 100C or even higher I would go with 70C for several hours (2-3) you are going to have to pilot this. That's what we do here for any sample has a tendency to lift or distort when retrieved (bone, skin). You may then be able to run the slides on a stainer but again if you see tissue detachment you are going to have to run them by hand.
We use a good superfrost plus slide such as histobond for bone and if we are running into problems with tissue lifting we let the unstained slides sit for a few days even a week before we stain and we find that that helps also. There is a human mitochondria antibody that we have used in the past in rat samples, but I can't remember what retrieval method works. There is also a human nuclear antibody which works well on frozen sections but can be tricky on paraffin, we have not had much success with it on paraffin sections. Good Luck Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC PO Box 18592 Boulder, Colorado 80308 office (303) 682-3949 fax (303) 682-9060 www.premierlab.com Ship to Address: 1567 Skyway Drive, Unit E Longmont, Colorado 80504 -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robin Dean Sent: Wednesday, December 08, 2010 9:20 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Reg: Beta-2 minroglobin IHC Hi all, We are trying to use beta-2 microglobulin IHC staining to ID human cells in goat joints that were engrafted with human cells some time ago and having nothing but trouble. Tissue was decalcified in immunodecal, formalin-fixed paraffin-embedded tissue. Beta-2 MG antibody is a polyclonal rabbit X human beta-2 antibody from Acris, and we are using DAKO Envision-HRP -con. secondary with DAB as chromogen. IgG isotype controls and DAB alone controls look clean. . Goat joints are falling off the charged slides with heat-induced citrate buffer antigen retrieval so went to enzymatic retrieval to keep them on the slide. o Does enzymatic antigen retrieval damage this epitope?? (previous successful uses of this antibody were with citrate antigen retrieval) o Any suggestions on how to keep sections on slides? Sections are kind of large, but pathologist wants that size. . Didn't get hardly any staining at 2.5 ug/ml of the beta-2MG antibody, but at 5 ug/ml everything stains including all of goat joints. . Are there other or better stains that are used for identifying human cells engrafted into other species (label uniformly most human cells)? I saw mention of some mitochondrial antgen antibodies but no specifics. . Does Beta-2 antibody label only human cells? Seems like there are homologous markers in other species that may label. Currently we aren't using any blocks except peroxidase block. Thought we might try a CAS block to see if that will prevent sticking? To non-human tissue. Any suggestions or help with these problems will be greatly appreciated. We have to get the stain to work and are running out of ideas. Robin Robin R. Dean, Ph.D. Senior Scientist & Study Director Comparative Biosciences, Inc. 786 Lucerne Dr. Sunnyvale, CA (408) 738-8060 robin_d...@compbio.com _______________________________________________ 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