Also, make sure that the block sits in the cryostat for a while to acclimate to the temperature. The cutting temp for lung (correct me if I'm wrong here) is probably about -20; the block should be the same temperature as the cryostat.
Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ________________________________ From: Colleen Forster <cfors...@umn.edu> To: Kim Merriam <kmerriam2...@yahoo.com> Sent: Monday, February 27, 2012 1:55 PM Subject: Re: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning Yep, the sample is too cold. Rubbing your finger across even with a glove (just linger a bit longer) will help alot. Colleen Forster U of MN On 2/27/2012 12:47 PM, Kim Merriam wrote: > As long as you don't need to use them for RNA analysis, the easiest thing to > do is just rub your finger (without glove) across the block (very briefly) > and then take the section. This will probably do the trick and hydrate it > just enough to make a difference. > > Kim > > Kim Merriam, MA, HT(ASCP)QIHC > Cambridge, MA > > > ________________________________ > From: "Kasai, Miki (NIH/NCI) [E]"<kas...@mail.nih.gov> > To: "histonet@lists.utsouthwestern.edu"<histonet@lists.utsouthwestern.edu> > Sent: Monday, February 27, 2012 1:34 PM > Subject: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning > > We are trouble-shooting cryosectioning mouse lung tissue. Often the lung > section tears or breaks apart during sectioning. In the past, if the lung > section is proving difficult to section, we take the OCT-embedded tissue and > re-embed it back into OCT (basically put fresh OCT into the original mold > and then place the OCT block with the tissue back into the mold such that > the exposed tissue is covered back with OCT). This is then placed back in > our -80°C. When sectioning the next day, the tissue is often easier to > section. > > One person in our lab tried to resolve the problem by brushing a little bit > of sterile water onto the tissue when sectioning. This appeared to hydrate > the tissue and it sectioned better. However, we weren't sure if this was a > good idea or not. Any feedback would be greatly appreciated. > > For background purposes our lung tissue are processed several ways: > > 1. Lungs are perfused with PBS, tissue extracted from mouse, placed in > PFA/sucrose for several hours and then embedded in OCT. > > 2. Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1), placed > in PFA/sucrose for several hours and then embedded in OCT. > > 3. Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1), > embedded in OCT and frozen by immersion into liquid nitrogen (just the > bottom half of the mold is lowered into LN). > > Much appreciation, > Miki Kasai > Biologist > Pediatric Oncology Branch > NCI, NIH > CRC, 1W Rm. 1-3-888 > 10 Center Drive > Bethesda, MD 20892 > (301) 496-2318 > > > _______________________________________________ > 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 > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
