Yes this all makes good sense and explains why I may not be able to have control of this because I do not always do the tissue fixation and processing myself. That first step of adequately fixing (cross linking the proteins) to protect them from tissue processing and AR are sooooo important. The one thing I can control is to make sure the slides are completely dry before baking them as Renee mentioned.
Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting, LLC 40864 Arkansas Ave Bennett, CO 80102 Fax: 720-859-4110 pru...@ihctech.net -----Original Message----- From: Gudrun Lang [mailto:gu.l...@gmx.at] Sent: Sunday, January 06, 2013 4:32 AM To: 'Patsy Ruegg' Cc: histonet@lists.utsouthwestern.edu Subject: AW: [Histonet] holly nuclei bat man I have a quiet semi-scientific explanation. Formalin-fixation stabilizes proteins via crosslinking. AR "solves" methylol-adducts due to formaldehyde and perhaps also some crosslinks. Nuclei also have a protein-scaffold, filled with nucleinacids. It seems like a "ladder" or "hole in tights". It seems like there is a kind of tension, after breaking a crosslink the hole "blubbs" out. This is also seen with pretreatment in hybridization-procedures. The more enzymatic and heat-retrieval the more holes. And there is a direct relationsship between fixation-quality and AR-strength. Bye Gudrun -----Ursprüngliche Nachricht----- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Patsy Ruegg Gesendet: Samstag, 05. Jänner 2013 21:38 An: histonet@lists.utsouthwestern.edu Betreff: [Histonet] holly nuclei bat man Happy New Year everyone! Can we start a discussion once again on what causes holes in nuclei? It is pretty clear in my experience it has something to do with heating at least for me with antigen retrieval. When I do HIER at ph 8 especially I am seeing a lot of holes in nuclei. H&E on same tissue alone without HIER/IHC doesn't seem to exhibit this artifact (holes in the nuclei). I bet it can also happen if the tissue is not fixed well enough to protect it from paraffin processing but right now I am seeing it more after HIER, don't see it as much on my IHC slides with no AR or when I use eier instead of hier. Also notice that ph6 HIER and Ph9 HIER are not as bad as when I use Ph8 for the same times and temps. Cheers, Patsy Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting, LLC 40864 Arkansas Ave Bennett, CO 80102 Fax: 720-859-4110 <mailto:pru...@ihctech.net> pru...@ihctech.net _______________________________________________ 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