[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
Re: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning
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
Re: [Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning
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.eduhistonet@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
