Just two things about crosslinking: 1- there is not such a thing as over crosslinkage, because when all the reaction sites have reacted, there are no reaction sites left to "overreact"; and 2- formaldehyde fixation is a reversible reaction and the tissue will lose the croslinkage even by placing them in distilled water instead of HIER (although it will take much more time). René J.
--- On Fri, 12/12/08, Merced Leiker <lei...@buffalo.edu> wrote: From: Merced Leiker <lei...@buffalo.edu> Subject: RE: [Histonet] Silly Question? To: pru...@ihctech.net Cc: "'histonet@lists.utsouthwestern.edu'" <histonet@lists.utsouthwestern.edu>, "'Pat Flannery'" <pjfne...@duke.edu> Date: Friday, December 12, 2008, 2:03 PM So I have a question about the cross-linking aspect of PFA...while I agree I need it to keep my epitope in place, is there such a thing as OVER-crosslinking (i.e., tissue spending TOO much time in formalin - weeks? months?) that would make my epitope difficult to near-impossible to retrieve? Loving the formaldehyde soap-boxes histonetters get onto... --On Friday, December 12, 2008 10:36 AM -0700 pru...@ihctech.net wrote: > > i have had this argument with the researchers at the University for 30 > years, somewhere back in the day they were told that commercially made > formalin had methanol in it (it does but just a little and does not hurt > anything in my experience) and that methanol would damage their tissue > for IHC, so they think they must use paraformaldehyde and make it fresh > themselves. Since new people make it all the time it often does not get > made up correctly and their stress over this issue is miss placed as they > should be using something commercial for consistancy and paying more > attention to adequate time for fixation in reg formalin. > Another anoying myth that is difficult to combat with them is that "we > should limit the fixation time in aldehyde fixatives because it will > cross link the proteins masking them for IHC", there fore i am always > getting tissue that has not been fixed long enough (at least 24 hrs. to > protect it from paraffin processing, because if the proteins are not > cross linked they can be alcohol fixed and/or washed away forever), the > people in research know about the cross linking fo aldehydes but do not > know that cross linking of proteins is a good thing and they also do not > know that we have advanced methods HIER or EIER for unmasking the > proteins, but we have no way of getting a protein back that has been lost > in processing because the sample was not adequately fixed. > > there i will get off my Friday soap box.................. > > Happy Holidays to all! > > Patsy > > > > -------- Original Message -------- > Subject: RE: [Histonet] Silly Question? > From: Merced Leiker <lei...@buffalo.edu> > Date: Fri, December 12, 2008 8:12 am > To: "Edwards, R.E." <r...@leicester.ac.uk>, 'Pat Flannery' > <pjfne...@duke.edu> > Cc: "'histonet@lists.utsouthwestern.edu'" > <histonet@lists.utsouthwestern.edu> > > In research lab situations particularly, one does not have the time or > technique for nailing down the ways of making each of the buffers, > reagents, and procedures work the "right" way or the most optimum way...a > lot of times it's students or postdocs just focused on getting their > project done and not caring how their fixative is made as long as it > "works" to some degree and, alas, it's up to us already over-booked > technicians to figure out the best way to make the PFA....and we usually > don't have a whole day (week, or year) to spend researching the > back-and-forth arguments, either! ;-) > > Merced > > --On Friday, December 12, 2008 2:04 PM +0000 "Edwards, R.E." > <r...@leicester.ac.uk> wrote: > >> You hit the nail on the head "That's what we always use", fear of >> change is a common human condition. >> >> -----Original Message----- >> From: histonet-boun...@lists.utsouthwestern.edu >> [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Pat >> Flannery Sent: 11 December 2008 16:59 >> To: histonet@lists.utsouthwestern.edu >> Subject: [Histonet] Silly Question? >> >> Please humor me on this if it's obvious (to everyone but me): why do >> we use paraformaldehyde (which is so inconvenient to make up) rather >> than buffered formalin or just diluted formaldehyde itself? >> >> It seems that around here, some folks prefer paraformaldehyde (either >> 2% or 4%) and others use formalin, while some others stick to diluted >> formaldehyde (I see all 4 on labels for specimens submitted for >> histology). Is it mostly a matter of personal preference or where you >> were trained (i.e. force of habit) or is there a valid reason to use >> each solution (basically the same chemical once in solution, merely >> buffered or not)? The only answer I've gotten when I've asked is, >> "That's what we always use." >> >> Thanks. >> >> -Pat Flannery (not a "real" histologist - I just play one in the lab) >> >> >> _______________________________________________ >> 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 >> > > > > Merced M Leiker > Research Technician II > 354 BRB (pkgs) / 140 Farber Hall (letters) > School of Medicine and Biomedical Sciences > State University of New York at Buffalo > 3435 Main St, Buffalo, NY 14214 > Ph: (716) 829-6033 > Fx: (716) 829-2725 > > "Without my flaws I'm really very boring." > - random internet blog commentator > > > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > Merced M Leiker Research Technician II 354 BRB (pkgs) / 140 Farber Hall (letters) School of Medicine and Biomedical Sciences State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 Ph: (716) 829-6033 Fx: (716) 829-2725 "Without my flaws I'm really very boring." - random internet blog commentator _______________________________________________ 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