Re: [Histonet] Bone samples
Ion Exchange Decal containers will change your experience forever. -Original Message- From: Gudrun Lang via Histonet Sent: Friday, January 26, 2024 3:32 AM To: 'Chakib Boussahmain' Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Bone samples Hi, In my opinion the hardness of the decalcified blocks is often rather due to the paraffin-processing than the residual calcium. Especially when the tissue is decalcified really long. The hardness comes from the dehydration and "cooking" of collagen fibers. So additional decal will not help reducing the calcium, but helps to reintroduce water into the collagen-grid. And this is helpful for softening and cutting. For myself, I often scratch the paraffin on the blocksurface away to face the bone directly to the water. Then I let them "swim" on my waterbath, until the surface is turned rather milky. After cooling again I cut in very very small steps to trim the surface. Sometimes it needs repeated swimming and cooling (and patience) to get a rather acceptable section. It is advantageous to pick them up on adhesive slides and let them dry in an 60°C oven to get rid of any residual water under the section. Hope this helps Gudrun -Ursprüngliche Nachricht- Von: Chakib Boussahmain via Histonet [mailto:histonet@lists.utsouthwestern.edu] Gesendet: Mittwoch, 24. Jänner 2024 23:34 An: histonet@lists.utsouthwestern.edu Betreff: [Histonet] Bone samples Hi guys, I hope this messagefinds you well. I am currently working on a study involving bone samples thathave been treated with slow decal and embedded in paraffin. I am facingchallenges in obtaining nice sections, and I was wondering if you could providesome guidance or recommendations. Thank you in advance for your help! Chakib ___ Histonet mailing list Histonet@lists.utsouthwestern.edu https://urldefense.com/v3/__http://lists.utsouthwestern.edu/mailman/listinfo/histonet__;!!NHLzug!Jh6cF0ugJABQPV4zi-V-Y0UPbZ4bUroLeBNYiAxwTdz6uiNtFvuO5RlsEtEwCqK5OzDLLv9GdeiF4tdq52dWnRiMVbxh3w$ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu https://urldefense.com/v3/__http://lists.utsouthwestern.edu/mailman/listinfo/histonet__;!!NHLzug!Jh6cF0ugJABQPV4zi-V-Y0UPbZ4bUroLeBNYiAxwTdz6uiNtFvuO5RlsEtEwCqK5OzDLLv9GdeiF4tdq52dWnRiMVbxh3w$ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Bone samples
Hi, In my opinion the hardness of the decalcified blocks is often rather due to the paraffin-processing than the residual calcium. Especially when the tissue is decalcified really long. The hardness comes from the dehydration and "cooking" of collagen fibers. So additional decal will not help reducing the calcium, but helps to reintroduce water into the collagen-grid. And this is helpful for softening and cutting. For myself, I often scratch the paraffin on the blocksurface away to face the bone directly to the water. Then I let them "swim" on my waterbath, until the surface is turned rather milky. After cooling again I cut in very very small steps to trim the surface. Sometimes it needs repeated swimming and cooling (and patience) to get a rather acceptable section. It is advantageous to pick them up on adhesive slides and let them dry in an 60°C oven to get rid of any residual water under the section. Hope this helps Gudrun -Ursprüngliche Nachricht- Von: Chakib Boussahmain via Histonet [mailto:histonet@lists.utsouthwestern.edu] Gesendet: Mittwoch, 24. Jänner 2024 23:34 An: histonet@lists.utsouthwestern.edu Betreff: [Histonet] Bone samples Hi guys, I hope this messagefinds you well. I am currently working on a study involving bone samples thathave been treated with slow decal and embedded in paraffin. I am facingchallenges in obtaining nice sections, and I was wondering if you could providesome guidance or recommendations. Thank you in advance for your help! Chakib ___ 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] Bone samples
Embed the bones diagonally in your molds if you're able (size depending) as this will allow for the greatest amount of paraffin support. Trim them very slowly, and if need be, place the blocks into the same "slow decal" solution for additional amount of time sufficient to enable better sectioning. Start checking in half hour or 45 minute intervals; rinse the blocks well in running water and attempt to section. If they're not ready, back into decal solution they go. I feel that very wet ice helps to facilitate sectioning better than ice that is drier and fresh out of the freezer. Just be sure to blot the face of the block with gauze before attempting to cut. There's my two cents! Thanks, Brian D. Cooper, HT (ASCP)CMQIHCCM| Histology Supervisor Department of Pathology and Laboratory Medicine Children's Hospital Los Angeles 4650 Sunset Blvd MS#43- Los Angeles, CA 90027 Ph: 323.361.3357 bcoo...@chla.usc.edu -Original Message- From: Chakib Boussahmain via Histonet Sent: Wednesday, January 24, 2024 2:34 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Bone samples (EXTERNAL EMAIL) CAUTION: BE CAREFUL WITH THIS MESSAGE* This email came from outside CHLA. Do not open attachments, click on links, or respond unless you expected this message and recognize the email address: histonet-boun...@lists.utsouthwestern.edu. Hi guys, I hope this messagefinds you well. I am currently working on a study involving bone samples thathave been treated with slow decal and embedded in paraffin. I am facingchallenges in obtaining nice sections, and I was wondering if you could providesome guidance or recommendations. Thank you in advance for your help! Chakib ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://secure-web.cisco.com/1oOSMT1FuuHaA4EXxjY3i9nIAlLf91ZjdN5e2afysmGGJYW7stpUWSe1_bubEbbdKpoHohJt-UQ7lkDBZ_BwXbYaFsVfbmyQtwDLG4qorqorzByzCfrpkWZnOwj1ZW6oWKhNoWYgB3KojnS_P3jfgs8QItdqAV5QGAqwEewtholWiGRWxd-ggJXBstwd3TKwVx9EdGpjtjB5T3-MrhwB6gYGfUkRWiSu2yt6PL7FbWYjl11MtUyGiXP6EcnRSb4fQZzykJavCYeY0XmEMR6pe-rxP8W-MsmYb7NizNBFhqYFhAbuqVDhn5joQziAtv9KSW6AAMi1oI6aQW2ncTolPPVcXljMP586zzPK9P-9m9eF_j2JwX1WZjXV7N5Du2QCgAl5Rsz3xpqP0b9vzEXZqZ021FHX5HNuoA0X0iKq9tyKn6gc_8OwLotyO2g4bQTZa_FQuxStxGkGG1wOCjwntTrbvdllnoHTF4aKGZMFPiA0/http%3A%2F%2Flists.utsouthwestern.edu%2Fmailman%2Flistinfo%2Fhistonet CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential or legally privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of this original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Bone samples long-term storage in 10% formalin or 4% paraformaldehyde
Dear Orla, Post fixation, we have stored our bone specimens in 1x PBS while having them sent out for MicroCT analysis. We have also stored them in 1x PBS at 4°C post fixation when necessary until further processing without having adverse affects on our staining. However, we chose conventional staining over IHC for our results. Best regards, ~Sean McBride Scientific Specialist Bone Tissue Engineering Center Carnegie Mellon Research Institute Suite 4311 700 Technology Drive Pittsburgh, PA 15219-3124 412-268-8275 (o) 412-915-1683 (m) 412-268-8275 (fax) smcbr...@andrew.cmu.edu On Dec 9, 2013, at 8:44 AM, Orla M Gallagher wrote: Thanks to everyone for your comments. I may not have been clear in my question - our researchers don't wish to decalcify these formalin-fixed bones yet, but rather to store them for more than a couple of weeks, in case they need to carry out MicroCT followed by histology later. I'm aware that the formalin or paraformaldehyde will degrade over time, but I just wondered if anyone has a protocol for storage without decalcification? I guess transfer to 70% ethanol is an option but this is also not ideal for longterm storage, and would need to be removed before decal in EDTA. All the best, Orla On 6 December 2013 16:12, Wineman, Terra terra.wine...@novusint.com wrote: I would suggest a different protocol if the tissue will not be processed for a while. I would say a week in 10%NBF and then transfer the bones to an EDTA decal solution. The bones will decal slowly without the affects of the formic acid. I am in research and this is what we do with our bones. Terra Wineman, HTL (ASCP)CM Research Biologist 636-926-7476 phone terra.wine...@novusint.com -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto: histonet-boun...@lists.utsouthwestern.edu] On Behalf Of pru...@ihctech.net Sent: Thursday, December 05, 2013 2:50 PM To: gu.l...@gmx.at; 'Orla M Gallagher' Cc: histonet@lists.utsouthwestern.edu Subject: RE: AW: [Histonet] Bone samples long-term storage in 10% formalin or 4% paraformaldehyde i would think u are correct in advising formic acid decal and then processing into paraffin for the best protection of the trap enzyme, immunoreactivity, etc. A couple of weeks in formalin should be fine. Paraformaldehyde show be the same as formalin. I do know a way to restore the enzyme activity for TRAP that may have been lost so if u need that let me know. - Original Message - Subject: AW: [Histonet] Bone samples long-term storage in 10% formalin or 4% paraformaldehyde From: Gudrun Lang gu.l...@gmx.at Date: 12/5/13 11:42 am To: 'Orla M Gallagher' o.m.gallag...@sheffield.ac.uk Cc: histonet@lists.utsouthwestern.edu Paraformaldehyd is formaldehyd in solid form. Formalin is the aequous solution of formaldehyd. So the main characteristics are the same. Gudrun Lang -Urspruuml;ngliche Nachricht- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Orla M Gallagher Gesendet: Donnerstag, 05. Dezember 2013 19:31 An: histonet@lists.utsouthwestern.edu Betreff: [Histonet] Bone samples long-term storage in 10% formalin or 4% paraformaldehyde Dear Histonetters, What is your opinion on storing bone samples long-term (more than a couple of weeks) in 10% formalin? As I was taught, best practice has always been to fix only as long as necessary, depending on the size of the sample, then decalcify and process to wax, and I always stress this to everyone I advise. However, research colleagues sometimes wish to do histology on bone samples that have been stored for months ..or even years! As the formalin pH becomes more acidic, there is formalin pigment and the immunoreactivity and TRAP enzyme activity is diminished or destroyed during long fixation, is there any way of minimising this e.g. has anyone tried regularly replacing the old formalin with fresh buffered formalin, or storing formalin-fixed bones in any other medium? I'm also interested in how best to fix in 4% paraformaldehyde and whether the problems are the same with long-term storage. Thanks for your comments. All the best, Orla -- ** Ms. Orla Gallagher Bone Analysis Laboratory Mellanby Centre for Bone Research Department of Human Metabolism D Floor Medical School University of Sheffield Beech Hill Road Sheffield S10 2RX UK Website: http://mellanbycentre.dept.shef.ac.uk Tel: 0044114-2713337 (office) 0044114-2713174 (lab) E-Mail: o.m.gallag...@sheffield.ac.uk *STOP*: Do you really need to print this e-mail? *BE GREEN:* Keep it on the screen. *Times Higher Education University of the Year* Data protection and confidentiality: The information contained in this message or any appended documents may be privileged and confidential and is intended for
