Your method makes no sense! It looks like something informally passed along among students and technicians who have never read a book. Cryoprotection means preventing formation of ice crystals or, as it's usually done, minimizing their size. Sucrose is a cryoprotectant; the higher the concentration the better, but strong solutions penetrate the tissue slowly. For fixed specimens dissolve the sucrose in water. (For unfixed, use an isotonic phosphate buffer, but that's only if a day or so at 4C is OK for your needs. Very fast freezing may be needed, especially for muscle.) OCT is not a cryoprotectant; it's goop that surrounds the specimen and enters cracks and spaces but it does not penetrate. OCT serves to hold each section together during transfer from the cryostat's knife onto a glass slide or coverslip. For some more information (much of it derived from Histonet) have a look at the Biological Stain Commission's FAQ, at https://biologicalstaincommission.org/faqlist.htm#CRYPRO. John Kiernan https://www.schulich.uwo.ca/anatomy//people/faculty/emeriti/kiernan_john.html = = = ________________________________ From: Tyrone Genade via Histonet <histonet@lists.utsouthwestern.edu> Sent: November 30, 2023 9:46 PM To: histonet@lists.utsouthwestern.edu <histonet@lists.utsouthwestern.edu> Subject: [Histonet] O.C.T. what MW PVA and PEG?
Hello, I'm using a cryoprotection protocol that involves 3-stage cryoprotection of 15% glucose O/N, then 30% + 50% OCT O/N and then finally O/N in OCT. Compared to previous protocols this works very well -- even when cutting through eyes and lenses (which had previously given a lot of grief). My issue is that preparing the 30% + 50% OCT is a schlep. The OCT puts up a lot of resistance against mixing with the 60% sucrose. It would be much simpler if I could prepare 30% sucrose with powdered PVA and PEG. Does anyone know what MW polymers of PVA and PEG to use and what concentrations to approximate commercial (Scigen Tissue-Plus) OCT? Thanks Tyrone Genade Ph.D. Quillen College of Medicine ETSU Johnson City, TN _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ________________________________ From: Tyrone Genade via Histonet <histonet@lists.utsouthwestern.edu> Sent: November 30, 2023 9:46 PM To: histonet@lists.utsouthwestern.edu <histonet@lists.utsouthwestern.edu> Subject: [Histonet] O.C.T. what MW PVA and PEG? Hello, I'm using a cryoprotection protocol that involves 3-stage cryoprotection of 15% glucose O/N, then 30% + 50% OCT O/N and then finally O/N in OCT. Compared to previous protocols this works very well -- even when cutting through eyes and lenses (which had previously given a lot of grief). My issue is that preparing the 30% + 50% OCT is a schlep. The OCT puts up a lot of resistance against mixing with the 60% sucrose. It would be much simpler if I could prepare 30% sucrose with powdered PVA and PEG. Does anyone know what MW polymers of PVA and PEG to use and what concentrations to approximate commercial (Scigen Tissue-Plus) OCT? Thanks Tyrone Genade Ph.D. Quillen College of Medicine ETSU Johnson City, TN _______________________________________________ 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
