hi, for alpha -SMA I just put the slide with section into a plastic jar with citrate buffer put the jar into microwave, "HIGH" for 1 min and quickly cool down by adding cool distilled water then use 0.3% trition-PBS solution to soak the section for 10 min. works very well.
i can show u pictures if u want,. 2009-02-12 TF 发件人: Sarah Tarran 发送时间: 2009-02-11 12:19:58 收件人: histonet 抄送: 主题: [Histonet] Help with antigen unmaksing on frozen sections 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
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