hI, i AM using formalin-free Zince fixative made from Zinc salt & Tris buffer. Then I made frozen sections after dehydration in 30% sucrose... No CD31+ staining...
I also tried shock frozen, acetone fixed tissue, the antibody works in this condition. 2009-07-02 TF 发件人： gayle callis 发送时间： 2009-07-02 00:09:12 收件人： ti...@foxmail.com; 'Histonet' 抄送： 主题： RE: [Histonet] RE: mouse brain fixation Attn: Annette Featherstone When you say zinc fixative, which one are you talking about? Becksteads Zinc TRIS buffer made up with zinc salts and formalin free? Or zinc formalin. The Becksteads zinc salt fixative does work, as cited in several papers in the literature, and we tried it but preferred to use frozen sections, just faster overall. And if you overexpose the tissues to dehydrant, clearing agents and heat of paraffin, the sectioning can be worse with the Beckstead fixative. It makes the tissue dry and crunchy. IF you do not fix the tissues properly and totally, then the alcohols used in processing will finish the fixation, and that isn't going to contribute to good sectioning either. How you make it up is critical, and should be done according to the original publication or Nitta et al paper. Gayle M. Callis From: TF [mailto:ti...@foxmail.com] Sent: Tuesday, June 30, 2009 11:33 PM To: gayle callis; 'Histonet' Subject: Re: [Histonet] RE: mouse brain fixation Attn: Annette Featherstone I tried Zinc fixative I made myself. Does not work at all? And, the tissue quality is very bad. Any comments? 2009-07-01 TF 发件人： gayle callis 发送时间： 2009-06-30 23:57:35 收件人： 'Histonet' 抄送： 主题： [Histonet] RE: mouse brain fixation Attn: Annette Featherstone Annette, You did not say what immunos e.g. antigens you are trying to stain? Nor how you are doing the actual staining method? More information would help please. If you are trying to stain for murine CD markers or some other cellular antigens, there are not many that work after FFPE. If you need to know this information, go to SEROTEC and look at murine CD or other cellular markers, and see if the antibody will work with FFPE paraffin applications. BD Bioscience also has applications that work, tested, reported, IHC or frozen sections and found in their Technical Data Sheets for any given antibody. There are some other fixatives to try for CD and other cellular markers - SEROTEC often refers to PLP (paraformaldehyde-lysine-periodate with recipe found on web through IHCWorld or Immunoportal, Google the keywords) while BD Bioscience refers to Becksteads Formalin free Zn TRIS buffer fixative. If you need the recipe for that, I will be happy to send. Otherwise BD Bioscience would be happy to sell that to you but it is cheaper and easy to make up in the lab. Gayle M. Callis HTL(ASCP)HT,MT Bozeman MT You wrote: We have been working with mouse brains that were fixed in 10% formalin for one month. We are currently getting "no" staining with our immunos. Can this really be the problem in light of antigen retrieval methods? Annette Featherstone _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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