[Histonet] Improving wrinkles and bubbles in fixed-frozen mouse brain cryo-sections?
Dear List, I was cryo-sectioning fixed-frozen and fresh-frozen mouse brains, and found a lot of wrinkles and folding in the fixed-frozen sections, while the fresh-frozen sections were pretty smooth. Below are my sectioning conditions and observations. I would appreciate any advice on improving the fixed-frozen sections. Tissues: fixed-frozen mouse brains (OCT embedded), fresh-frozen mouse brains (OCT embedded). Tissue temperature: at -80 deg for at least 48hrs, equilibrated to -18 deg for 1hr before sectioning. Thickness: 14um Slide: Superfrost plus Cryostat chamber and head temperatures: -18 deg, -21 deg respectively. Observations: When the fresh-frozen sections came out of the block, they rolled up smoothly, “jumped” to the slide immediately, and unrolled themselves smoothly. But the fixed-frozen sections looked more “limp” – they were “wavy”, could not roll up, and when I put a slide over them, they could not extend smoothly thereby creating wrinkles, folding and even bubbles. It looked as if the fixed-frozen block was “warmer” than the fresh-frozen block, but in fact they had been equilibrated to -18 deg for the same amount of time (~1hr). And I believe it was also not a blade artifact, because I alternated between cutting fixed-frozen and fresh-frozen sections several times using the same blade. The fresh-frozen sections looked fine each time, but the fixed-frozen sections were always problematic. I also believe there was no temperature difference between the slides used for fixed- or fresh-frozen sections (both were stored at room temperature). My protocol for preparing the fixed-frozen tissues is as follows: 1. Transcardial perfusion of PBS (10ml) and 4% PFA (10ml). 2. Fixation with 4% PFA at 4°C overnight (~18hrs). 3. Wash with PBS (20min x 3). 4. Dehydration with 15% sucrose (in 1X PBS) at 4°C until tissue sinks (~12hrs). 5. Dehydration with 30% sucrose (in 1X PBS) at 4°C until tissue sinks (~24hrs). 6. Embedding in OCT with dry ice and 100% EtOH until OCT solidifies. 7. Stored at -80°C. What do you think I could do to improve the fixed-frozen sections? Your input would be much appreciated. Thank you, Anna Luo PhD Candidate Department of Neuroscience Johns Hopkins Medical Institutions ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] combining cytology and histology
Does anyone have a lab that has combined histology and cytology? I would like to know how they are staffed and if accessioning handles all samples or if they are handled separate? Best Wishes Pam ~ National Histology Professionals Day 3/10/21 Pathologists' Assistant Day 4/14/2021 Medical Laboratory Professionals Week April 18-24, 2021 National Cytotechnology Day 5/13/2021 +++ Pam Richardson Clinical Manager Gundersen Health System Laboratory Services Email: pkric...@gundersenhealth.org Phone: 608 775-4133 Fax: 608 775-6136 Interdepartmental Mail Stop: H04-007 E-visit us at: http://www.gundersenhealth.org -Original Message- From: histonet-requ...@lists.utsouthwestern.edu Sent: Friday, July 30, 2021 12:00 PM To: histonet@lists.utsouthwestern.edu Subject: Histonet Digest, Vol 212, Issue 17 Attention: You have received this email from someone outside of Gundersen. Please use caution responding, opening attachments or clicking on links. Report this email using the "Report Phish" button in Outlook if you are uncertain about it, if it asks for an account validation, password reset, or claims to be a security alert. Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit http://secure-web.cisco.com/1HnFoR0qyvMsrOykVjF7ey7PQPfwsd0GqzKyT00hu9jpwH6ocxUeggOndRLjea777X5fSBbxKjNQY5Vor1Bd6X1ARyTMIFE-BhXFF1zCSf1Ro-HPuM3YDnDyR19MATvctuhZREvijDaYsHtaPHhXyWS36Iz_WbFhHMmgNJxRFhHRcDv0DGyX8s1StrDgC_E7KGcALMbbz7fh-bH9zih2Ip_dCuXYKL8HDkeqQKc_yuLwqBMHeam4DAvihKBfD4NcRvBRsaI-FBImeqYbIFoviF_mQEQJTp5rT39p0irCcGB5_6C7H-w5-OmVn0p-JZSSE/http%3A%2F%2Flists.utsouthwestern.edu%2Fmailman%2Flistinfo%2Fhistonet or, via email, send a message with subject or body 'help' to histonet-requ...@lists.utsouthwestern.edu You can reach the person managing the list at histonet-ow...@lists.utsouthwestern.edu When replying, please edit your Subject line so it is more specific than "Re: Contents of Histonet digest..." ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Histonet Digest, Vol 213, Issue 6
We just recently purchased the Epredia NX50, which we use with our older HM525s. We love it, I just wish they would make height adjustability a standard option on them! Jennifer Wooten, BA, BS, HTL (ASCP)CM Pronouns: she/her/hers Technical Supervisor | Anatomic Pathology | University Hospital jennifer.woo...@tricore.org Desk: 505.272.5486 | Fax: 505.272.0240 TriCore Reference Laboratories 2211 Lomas Blvd NE Albuquerque, NM 87106 www.tricore.org -Original Message- From: histonet-requ...@lists.utsouthwestern.edu Sent: Monday, August 9, 2021 11:00 AM To: histonet@lists.utsouthwestern.edu Subject: Histonet Digest, Vol 213, Issue 6 Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit https://urldefense.com/v3/__http://lists.utsouthwestern.edu/mailman/listinfo/histonet__;!!NYT1fPLx!su441bkXoI6KEdpU9J6Y_kFnYpA0qht9oL1QCpqQA_eG5pBxEfSU0J4TDbSDdzaAJBCmQQ$ or, via email, send a message with subject or body 'help' to histonet-requ...@lists.utsouthwestern.edu You can reach the person managing the list at histonet-ow...@lists.utsouthwestern.edu When replying, please edit your Subject line so it is more specific than "Re: Contents of Histonet digest..." IMPORTANT: This message originates from TriCore Reference Laboratories or one of its affiliate organizations or representatives. Please note that the information contained in this e-mail message (including any attachments) should be considered confidential and intended for the use of the individual or entity named appropriately. If the reader of this message is any other than the individual named above or an agent responsible for delivery, you are hereby notified that any inappropriate dissemination, distribution, or copying of this communication is strictly prohibited. If you have received this communication in error, please notify the sender immediately. Any errant copy of this message must be deleted. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Wrinkles and bubbles in fixed-frozen mouse brain cryo-sections.
Dear List, I was cryo-sectioning fixed-frozen and fresh-frozen mouse brains, and found a lot of wrinkles and folding in the fixed-frozen sections, while the fresh-frozen sections were pretty smooth. Below are my sectioning conditions and observations. I would appreciate any advice on improving the fixed-frozen sections. Tissues: fixed-frozen mouse brains (OCT embedded), fresh-frozen mouse brains (OCT embedded). Tissue temperature: at -80 deg for at least 48hrs, equilibrated to -18 deg for 1hr before sectioning. Thickness: 14um Slide: Superfrost plus Cryostat chamber and head temperatures: -18 deg, -21 deg respectively. Observations: When the fresh-frozen sections came out of the block, they rolled up smoothly, “jumped” to the slide immediately, and unrolled themselves smoothly. But the fixed-frozen sections looked more “limp” – they were “wavy”, could not roll up, and when I put a slide over them, they could not extend smoothly thereby creating wrinkles, folding and even bubbles. I have attached an image showing my fresh-frozen sections at the top, and fixed-frozen at the bottom: https://drive.google.com/file/d/1FMWcW7g9N_yUrhsiEMe8orvR3vM32RiU/view?usp=sharing It looked as if the fixed-frozen block was “warmer” than the fresh-frozen block, but in fact they had been equilibrated to -18 deg for the same amount of time (~1hr). And I believe it was also not a blade artifact, because I alternated between cutting fixed-frozen and fresh-frozen sections several times using the same blade. The fresh-frozen sections looked fine each time, but the fixed-frozen sections were always problematic. I also believe there was no temperature difference between the slides used for fixed- or fresh-frozen sections (both were stored at room temperature). My protocol for preparing the fixed-frozen tissues is as follows: 1. Transcardial perfusion of PBS (10ml) and 4% PFA (10ml). 2. Fixation with 4% PFA at 4°C overnight (~18hrs). 3. Wash with PBS (20min x 3). 4. Dehydration with 15% sucrose (in 1X PBS) at 4°C until tissue sinks (~12hrs). 5. Dehydration with 30% sucrose (in 1X PBS) at 4°C until tissue sinks (~24hrs). 6. Embedding in OCT with dry ice and 100% EtOH until OCT solidifies. 7. Stored at -80°C. What do you think I could do to improve the fixed-frozen sections? Your input would be much appreciated. Thank you, Anna Luo PhD Candidate Department of Neuroscience Johns Hopkins Medical Institutions ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] cryostat
Hi Mehndi, Same here, I had been using Epredia cryostat for the last several years. I like it very much. A video on youtube shows its beautiful features. https://youtu.be/oMNt6RT5cWI Best, Haiyan On Mon, Aug 9, 2021 at 7:04 AM Morken, Timothy via Histonet < histonet@lists.utsouthwestern.edu> wrote: > Mehndi, we also cut frozens for DIF on kidney and muscle histochem. We > have two Epredia NX70 cryostats. We've used them for 8 years now and really > like them. > > Tim Morken > Supervisor, Electron Microscopy/Neuromuscular Special Studies > Department of Pathology > UC San Francisco Medical Center > > > -Original Message- > From: Histology via Histonet > Sent: Monday, August 09, 2021 6:01 AM > To: histonet@lists.utsouthwestern.edu > Subject: [Histonet] cryostat > > Hello, > > Trying to get everyone's opinion on cryostats. We have had the Leica > CM1850 forever and it is not being supported by Leica anymore. Thinking of > trying a different company maybe Tanner or Rankin. What does everyone use > and like. We do not cut Mohs, only DIFs. Maybe 1 a day. > > Thanks to all, > > Mehndi Helgren > > Dominion Pathology Laboratories > 733 Boush Street > Suite 200 > Norfolk, VA 23510 > 757-664-7901 > > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > > https://urldefense.com/v3/__http://lists.utsouthwestern.edu/mailman/listinfo/histonet__;!!LQC6Cpwp!8z84Bh8A8kSfaOqndOJm4QoOseISNw1HiCiz-OksuM_qvkfAVIH4wUOs3iM9HGD5XluxSg$ > > ___ > 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] cryostat
Mehndi, we also cut frozens for DIF on kidney and muscle histochem. We have two Epredia NX70 cryostats. We've used them for 8 years now and really like them. Tim Morken Supervisor, Electron Microscopy/Neuromuscular Special Studies Department of Pathology UC San Francisco Medical Center -Original Message- From: Histology via Histonet Sent: Monday, August 09, 2021 6:01 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] cryostat Hello, Trying to get everyone's opinion on cryostats. We have had the Leica CM1850 forever and it is not being supported by Leica anymore. Thinking of trying a different company maybe Tanner or Rankin. What does everyone use and like. We do not cut Mohs, only DIFs. Maybe 1 a day. Thanks to all, Mehndi Helgren Dominion Pathology Laboratories 733 Boush Street Suite 200 Norfolk, VA 23510 757-664-7901 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu https://urldefense.com/v3/__http://lists.utsouthwestern.edu/mailman/listinfo/histonet__;!!LQC6Cpwp!8z84Bh8A8kSfaOqndOJm4QoOseISNw1HiCiz-OksuM_qvkfAVIH4wUOs3iM9HGD5XluxSg$ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] cryostat
Hello, Trying to get everyone's opinion on cryostats. We have had the Leica CM1850 forever and it is not being supported by Leica anymore. Thinking of trying a different company maybe Tanner or Rankin. What does everyone use and like. We do not cut Mohs, only DIFs. Maybe 1 a day. Thanks to all, Mehndi Helgren Dominion Pathology Laboratories 733 Boush Street Suite 200 Norfolk, VA 23510 757-664-7901 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
