[Histonet] Cryosectioning undecalcified bone
Dear Histonetters, We would like to develop cryosectioning for undecalcified mouse and rat bones. Previous attempts to use the CryoJane system from Instrumedics with an old Bright cryostat and solid tungsten carbide blade a few years ago didn't result in successful reproducible sections - marrow without bone or bone without marrow on the slide, in spite of various freezing and prep. protocols used by my colleague. I suspect the old cryostat and blade weren't helping either. Have you any recommendations on the best cryostat to use to do this? We'd like to also use the cryostat for standard soft tissue sectioning. I've seen Leica mentioned in a few papers relating to CryoJane. There appears also to be a tape transfer method from Kawamoto's Section-Lab Co. Ltd. (i...@section-lab.jp) Does anyone use this method or know whether the consumables are still available for purchase, as the website seems dormant? Thanks for any advice, 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. http://www.shef.ac.uk/faculty/medicine-dentistry-health/2.9274/emailwording http://www.sheffield.ac.uk/humanmetabolism/greenimpact http://www.shef.ac.uk/faculty/medicine-dentistry-health/2.9274/emailwording http://www.sheffield.ac.uk/visitors/mapsandtravel ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Fixation in concentrated 37% formaldehyde
Dear Histonetters, What would the effect be of fixing tissues samples in concentrated formaldehyde instead of 10% buffered formalin? One of our researchers would like us to prepare some bones for histology staining which have been fixed in 37% formaldehyde by mistake and stored for 4 years. I assume there will be formalin pigment in these samples, so even a HE may not look great, while any enzyme histochemistry or immunohistochemistry is probably not worth trying. I haven't seen the samples yet as they will be arriving from another UK university. Has anyone out there fixed any tissues in concentrated formaldehyde by accident or by design? Thanks, 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. http://www.shef.ac.uk/faculty/medicine-dentistry-health/2.9274/emailwording http://www.sheffield.ac.uk/visitors/mapsandtravel ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: AW: [Histonet] Bone samples long-term storage in 10% formalin or 4% paraformaldehyde
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 the exclusive use of the addressee(s). If you are not the addressee, any disclosure, reproduction, distributions, other dissemination or use of this message is strictly prohibited and may be unlawful. If you receive this correspondence in error please contact the sender immediately and permanently delete/destroy what you have received. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http
[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 the exclusive use of the addressee(s). If you are not the addressee, any disclosure, reproduction, distributions, other dissemination or use of this message is strictly prohibited and may be unlawful. If you receive this correspondence in error please contact the sender immediately and permanently delete/destroy what you have received. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] How to remove 80% ethanol from bones before decalcifying in EDTA
Dear Histonetters, I have some mouse bones samples which have been fixed in 70% ethanol for a few weeks and stored in 80% ethanol for a week; now our colleague would like to decalcify them in EDTA for wax embedding and staining. What is the best way to remove the ethanol before transfer to EDTA? Thanks, 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 the exclusive use of the addressee(s). If you are not the addressee, any disclosure, reproduction, distributions, other dissemination or use of this message is strictly prohibited and may be unlawful. If you receive this correspondence in error please contact the sender immediately and permanently delete/destroy what you have received. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Bone decalcifying/processing problem
Dear Histonetters, Has anyone ever seen large holes/vacuoles in the bone marrow of mouse long bones after decalcification and processing to wax? We've had this problem with a study which was fixed in 4% paraformaldehyde and decalcified at 4 degrees C over a 14 day period according to a routinely-used protocol involving changes of 15% EDTA/0.5% PFA in PBS pH 8.0, rinsed in PBS to remove EDTA before processing to paraffin wax. The marrow is filled with large holes, almost like balloons. I'm happy to send images to anyone who might be able to help! Best wishes, 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 the exclusive use of the addressee(s). If you are not the addressee, any disclosure, reproduction, distributions, other dissemination or use of this message is strictly prohibited and may be unlawful. If you receive this correspondence in error please contact the sender immediately and permanently delete/destroy what you have received. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Maximum bone sample size for methyl methacrylate embedding
Dear Histonetters, Would anyone advise on the maximum size sample of undecalcified bone which could be properly processed into methyl methacrylate for sectioning and staining for Goldner's trichrome? Would anyone have a protocol for processing large bone samples (possibly 2 x1cm) into MMA as most of the protocols I've seen are based on Bordier trephine 5mm iliac crest biopsies. Thank you, Orla -- ** Ms. Orla Gallagher Bone Analysis Laboratory Mellanby Centre for Bone Research D Floor Medical School University of Sheffield Beech Hill Road Sheffield S10 2RX Website: http://mellanbycentre.dept.shef.ac.uk Tel: 00353114-2713337 (office) 00353114-2713174 (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 the exclusive use of the addressee(s). If you are not the addressee, any disclosure, reproduction, distributions, other dissemination or use of this message is strictly prohibited and may be unlawful. If you receive this correspondence in error please contact the sender immediately and permanently delete/destroy what you have received. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] immunofluorescence on methyl methacrylate embedded human bone samples
Dear Histonetters, Would there be a problem with autofluorescence in bone when performing immunofluorescence on formalin-fixed methyl methacrylate-embedded human bone marrow trephines? Thanks, Orla -- ** Ms. Orla Gallagher Bone Analysis Laboratory Mellanby Centre for Bone Research D Floor Medical School University of Sheffield Beech Hill Road Sheffield S10 2RX Website: http://mellanbycentre.dept.shef.ac.uk Tel: 00353114-2713337 (office) 00353114-2713174 (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 the exclusive use of the addressee(s). If you are not the addressee, any disclosure, reproduction, distributions, other dissemination or use of this message is strictly prohibited and may be unlawful. If you receive this correspondence in error please contact the sender immediately and permanently delete/destroy what you have received. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] EDTA decalcification of bones which have been fixed in 70% ethanol
Dear Histonetters, We would like to decalcify some mouse bones in EDTA pH 7 which have been received by our lab already fixed directly in 70% ethanol rather than in formalin or paraformaldehyde. How would you recommend preparing these bones for decalcification e.g. whether to post-fix in formalin or to wash out the ethanol before transferrring to EDTA? I realise that ethanol is not the best fixative to use, especially as the end user may want to do immunocytochemistry or enzyme histochemistry using TRAP. Thanks, Orla -- ** Ms. Orla Gallagher Bone Analysis Laboratory Mellanby Centre for Bone Research D Floor Medical School University of Sheffield Beech Hill Road Sheffield S10 2RX Website: http://mellanbycentre.dept.shef.ac.uk Tel: 0114-2713337 (office) 0114-2713174 (lab) E-Mail:o.m.gallag...@sheffield.ac.uk Please think about the environment before printing this email ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
