RE: [Histonet] Bone decalcifying/processing problem
Orla, You seem to have introduced many poor histologic processing practices into your technique. Firstly ???4% paraformaldehyde. How did you make this up? The nomenclature is incorrect (see Histonet archives and Manoonkitiwongsa PS, Schultz RL. (2002) Proper nomenclature of formaldehyde and paraformaldehyde fixatives for Histochemistry Histochem J 34: 365-367). Secondly, why fix and decal at 4oC. Low temperatures slow down the rate of diffusion and therefore fixation and can cause the formation of ice crystal artefact, which seems like what you have here. Also if you are going to decalcify tissues then I would recommend doubling your fixation time. Remember one of the big advantages of aldehyde fixation is that it cross-links and thereby protects cell and tissue constituents from processing. Try doing it all at room temperature, I bet that the artefacts will disappear. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Orla M Gallagher Sent: Saturday, 13 July 2013 12:52 AM To: histonet@lists.utsouthwestern.edu Subject: [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 * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Sydney Children's Hospitals Network. This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Sydney Childrens Hospital's Network accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ 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
RE: [Histonet] Bone decalcifying/processing problem
Orla I know this might sound silly but normal mouse bone marrow contains both fat and hemoatopoietic marrow, the areas that are fat will appear like round voids. Is that what you are seeing? Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 881-0763 cell (303) 682-9060 fax l...@premierlab.com Ship to address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Orla M Gallagher [o.m.gallag...@sheffield.ac.uk] Sent: Friday, July 12, 2013 8:52 AM To: histonet@lists.utsouthwestern.edu Subject: [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 mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
