[Histonet] Frozen tissue detachment
Hi everyone, We're having trouble with frozen tissue detachment (mouse aortic root). Our procedure is as follows: 1. Fresh heart into NBF 4% (Lilly's) 24h, fridge (+4) 2. OCT (tissue-tek) 2h, fridge (+4) 3. Quickly frozen in icecold isopentan 4. Freezer (-20) until sectioned at 10 µm in cryostat (at approx -20) 5. RT one to several hours before being put back into the freezer. We use Superfrost glass slides. The *only* thing I can think of is wrong, is that the sections have been thawed/frozen several times before being used for staining (we do that to find the best section for our stainings) -do you think that's the problem? Another thing could be that after being sectioned we don't have a specific amount of time and temperature at which the sections should dry.. I found out recently regarding my IHC protocol, that removing the OCT with 70% EtOH (10 min = dry 20 min) makes the tissue stick better than removing it with dH2O or TBS..but then the specific staining also became much more faded..any ideas what else to do? Sorry about all the questions -I'm relatively new to this field, but find it very interesting. Best, Marie ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
AW: [Histonet] microtomy
I thought it as a rule, that the thickness of slides vary depending on everything (inclusive weather, humidity, person, speed, temperature, cutting angle...). Although not very scientific, only the techs experience and eye will fix the problem. As for the duration on ice: If you have wet ice, the tissue can take up the humidiy and swell (if too long, the tissue comes beyond the surface), but the slides wouldn't be thicker. The cooler the block, the longer the temperature is held while sectioning, the longer the thickness is stable. But if you give one breath to the surface, the section is possibly double thick. Gudrun -Ursprüngliche Nachricht- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Webb, Dorothy L Gesendet: Freitag, 02. März 2012 19:07 An: 'histonet@lists.utsouthwestern.edu' Betreff: [Histonet] microtomy We have run into an interesting scenario and wondering what the experts think! We cut bone marrow bx's and lymph nodes for lymphoma @ 3 microns on one particular microtome. Within the past month, the hematopathologist has felt the sections are thicker than the usual 3 microns. I had our service technician measure the microns and the equipment was cutting as set. I had the blocks cut on a different microtome and we have seen variations there also. My question is, does the amount of time on ice make a minor difference in the section thickness? I know a lot of responses may be the difference in the tech cutting inasmuch as how fast they turn the rotations, etc., but,we have ruled out that variable by having more than one tech cut at the microtome in question. I am stymied as to how to remedy this fluctuation! This is why we love histology, so many variables to create a problem and why I love histonet, so many techs to help one through a dilemma!! Thank you!! Dorothy Webb, HT (ASCP) This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ 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: AW: [Histonet] microtomy
Correct Gudrun! Where is the like button? Paula K. Pierce, HTL(ASCP)HT President Excalibur Pathology, Inc. 8901 S. Santa Fe, Suite G Oklahoma City, OK 73139 405-759-3953 Lab 405-759-7513 Fax www.excaliburpathology.com From: Gudrun Lang gu.l...@gmx.at To: 'Webb, Dorothy L' dorothy.l.w...@healthpartners.com Cc: histonet@lists.utsouthwestern.edu Sent: Saturday, March 3, 2012 7:39 AM Subject: AW: [Histonet] microtomy I thought it as a rule, that the thickness of slides vary depending on everything (inclusive weather, humidity, person, speed, temperature, cutting angle...). Although not very scientific, only the techs experience and eye will fix the problem. As for the duration on ice: If you have wet ice, the tissue can take up the humidiy and swell (if too long, the tissue comes beyond the surface), but the slides wouldn't be thicker. The cooler the block, the longer the temperature is held while sectioning, the longer the thickness is stable. But if you give one breath to the surface, the section is possibly double thick. Gudrun -Ursprüngliche Nachricht- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Webb, Dorothy L Gesendet: Freitag, 02. März 2012 19:07 An: 'histonet@lists.utsouthwestern.edu' Betreff: [Histonet] microtomy We have run into an interesting scenario and wondering what the experts think! We cut bone marrow bx's and lymph nodes for lymphoma @ 3 microns on one particular microtome. Within the past month, the hematopathologist has felt the sections are thicker than the usual 3 microns. I had our service technician measure the microns and the equipment was cutting as set. I had the blocks cut on a different microtome and we have seen variations there also. My question is, does the amount of time on ice make a minor difference in the section thickness? I know a lot of responses may be the difference in the tech cutting inasmuch as how fast they turn the rotations, etc., but,we have ruled out that variable by having more than one tech cut at the microtome in question. I am stymied as to how to remedy this fluctuation! This is why we love histology, so many variables to create a problem and why I love histonet, so many techs to help one through a dilemma!! Thank you!! Dorothy Webb, HT (ASCP) This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ 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
RE: [Histonet] lab. setting up
Dear Mohamed, Please feel free to email me directly. I will gladly assist you. I am currently opening a facility in California, and would be pleased to share with you the equipment decisions we made, and why. Most equipment is made in Germany, England or Japan. As such, I am sure it is all very available in Egypt. Let me know if I can be of help. Sent from my Windows Phone From: mohamed abd el razik Sent: 3/2/2012 12:59 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] lab. setting up dear all on histonet I'm going to setup a new histology lab. in Egypt. I need to contact with you to advice me about the best and comfotable facilities microtomes- processors-automatic stainers and so on . Mohamed Ass. Lec. of histology Faculty of Vet. Med. Cairo University Egypt ___ 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: AW: [Histonet] microtomy
We are talking about reality here. Not what it says in some text book written by someone who hasn't cut a block in years. The tech is going to take the first good looking section they get after they have sat on the ice most Likely waiting for the tech who is so busy with 100 other task. So if long sitting bloated blocks is a new practice in your daily routine. Sure. You can get some thick sections tossed in your days work. Happy days. Sent from my iPhone On Mar 3, 2012, at 8:39 AM, Gudrun Lang gu.l...@gmx.at wrote: I thought it as a rule, that the thickness of slides vary depending on everything (inclusive weather, humidity, person, speed, temperature, cutting angle...). Although not very scientific, only the techs experience and eye will fix the problem. As for the duration on ice: If you have wet ice, the tissue can take up the humidiy and swell (if too long, the tissue comes beyond the surface), but the slides wouldn't be thicker. The cooler the block, the longer the temperature is held while sectioning, the longer the thickness is stable. But if you give one breath to the surface, the section is possibly double thick. Gudrun -Ursprüngliche Nachricht- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Webb, Dorothy L Gesendet: Freitag, 02. März 2012 19:07 An: 'histonet@lists.utsouthwestern.edu' Betreff: [Histonet] microtomy We have run into an interesting scenario and wondering what the experts think! We cut bone marrow bx's and lymph nodes for lymphoma @ 3 microns on one particular microtome. Within the past month, the hematopathologist has felt the sections are thicker than the usual 3 microns. I had our service technician measure the microns and the equipment was cutting as set. I had the blocks cut on a different microtome and we have seen variations there also. My question is, does the amount of time on ice make a minor difference in the section thickness? I know a lot of responses may be the difference in the tech cutting inasmuch as how fast they turn the rotations, etc., but,we have ruled out that variable by having more than one tech cut at the microtome in question. I am stymied as to how to remedy this fluctuation! This is why we love histology, so many variables to create a problem and why I love histonet, so many techs to help one through a dilemma!! Thank you!! Dorothy Webb, HT (ASCP) This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ 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
