Hi everyone, I some help with antigen on frozen sections as I am having a lot of trouble getting antibodies to work. I am using a GFP mouse so can't formalin fix, parafin embed my samples. I tried frsh freezing but found that the GFP leaked so I couldn't use that either. So I currently fix my tissue in 4% paraformaldehyde for 2 hours, then cryopreserve in 18% sucrose overnight before embedding in OCT the following day. I have tried non-enzymatic heat retieval (boiling for 15 minutes in Dako's Target retrieval solution pH 9) but this destroys the GFP, if I use proteinase K it 'eats' away at the tissue. I have tried diluting it but that hasn't helped get the antibodies to work. I have also tried overnight at 4 degree incubations without any luck.
Does anyone have any other ideas that I can try to get the antibodies to work? The antibodies that I am having problems with are: alpha smooth muscle actin, LSP-1, procollagen, serum amyloid p and TGF-beta. Thanks Sarah Tarran Postdoctoral Fellow Vascular Biology Research Centre, Department of Surgery Westmead Hospital, Westmead, NSW, 2145 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
