Re: [Histonet] need tips for cross-sectioning of cortical bone
Why not embed in resin (MMA) and take thicker sections and then grind/ polish them down? If you went this route, you could then use flourescent labels and quantify mineral apposition rate and bone formation rate. Let me know if you are interested. I can help you get started and direct you to low cost equipment options. Jack On Apr 22, 2010, at 9:58 AM, Connolly, Brett M brett_conno...@merck.com wrote: A colleague is having trouble getting wrinkle-free sections of decalcified, paraffin embedded femur. Any tips?? Thanks, Brett M. Connolly, Ph.D. Molecular Imaging Team Leader Merck Co., Inc. PO Box 4, WP-44K West Point, PA 19486 tel. 215-652-2501 fax. 215-993-6803 brett_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html ) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ 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] need tips for cross-sectioning of cortical bone
You did not say whether these are cross sections or mid sagittal? Cross sections are always tougher. Key is to make sure the cortical bone is well processed and infiltrated with a hard paraffin e.g. Tissue Prep 2 (Fisher Scientific ala Thermo Scientific). Try this old bonehead trick is cut tiny V-shaped notches with razor blade or used microtome blade on the sides of block, parallel to the blade. You may have to do this top and bottom too. Take care to NOT make these notches huge. This permits the paraffin of each section in ribbon to expand, relax onto the water bath along with the bone section. Another trick is try laying section on RT 5 to 10% alcohol, pick up on slide, then go to warm water bath, lower section slowly to flattening. The key here is to NOT let upper part of paraffin of a section totally release from slide while going into warm water (at an angle) in other words, the section flattens while paraffin portion is still attached to slide during flattening. Also, change the blade frequently. Sharpest possible edge helps, and hopefully high profile which is more stable than low profile for decalcified bone microtomy. Good luck Gayle M. Callis HTL/HT/MT(ASCP) -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jack Ratliff Sent: Tuesday, May 11, 2010 5:03 AM To: Connolly, Brett M Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] need tips for cross-sectioning of cortical bone Why not embed in resin (MMA) and take thicker sections and then grind/ polish them down? If you went this route, you could then use flourescent labels and quantify mineral apposition rate and bone formation rate. Let me know if you are interested. I can help you get started and direct you to low cost equipment options. Jack On Apr 22, 2010, at 9:58 AM, Connolly, Brett M brett_conno...@merck.com wrote: A colleague is having trouble getting wrinkle-free sections of decalcified, paraffin embedded femur. Any tips?? Thanks, Brett M. Connolly, Ph.D. Molecular Imaging Team Leader Merck Co., Inc. PO Box 4, WP-44K West Point, PA 19486 tel. 215-652-2501 fax. 215-993-6803 brett_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html ) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ 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 __ Information from ESET Smart Security, version of virus signature database 5103 (20100510) __ The message was checked by ESET Smart Security. http://www.eset.com __ Information from ESET Smart Security, version of virus signature database 5103 (20100510) __ The message was checked by ESET Smart Security. http://www.eset.com __ Information from ESET Smart Security, version of virus signature database 5105 (20100511) __ The message was checked by ESET Smart Security. http://www.eset.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] need tips for cross-sectioning of cortical bone
Brett- Most wrinkles in decalcified bone sections come from stretching of the decalified bone that occurs during the sectioning process. I would suggest a rather simple solution. Allowing the sections to flatten on the waterbath might take longer or a higher temperature. If paraffin surrounding the bone section seems to be containing it, not allowing it to expand to eliminate the wrinkles, gently tease it off. After all, you want the bone section, not the paraffin. A room temperature water bath (or 30% EtOH) to lay out the sections on to tease out any wrinkles before transfering the sections to the warm waterbath may also help. I hope this helps! Joe Saby, BA HT From: Adam . anonwu...@gmail.com To: Connolly, Brett M brett_conno...@merck.com Cc: histonet@lists.utsouthwestern.edu Sent: Thu, April 22, 2010 11:12:26 AM Subject: Re: [Histonet] need tips for cross-sectioning of cortical bone Cutting bone is very hard, and I'm by no means an expert at it. Assuming the blocks are properly fixed and decalcified, the best thing I've found is to put the blocks at -20C for 5-10 mins to cool them, then right before you cut them, rub a little ice water on the face of the block. That should help you get some nice clean cuts. If the sections become hard to cut again, reapply the ice water. If that stops working, back in the freezer they go. Adam On Thu, Apr 22, 2010 at 9:58 AM, Connolly, Brett M brett_conno...@merck.com wrote: A colleague is having trouble getting wrinkle-free sections of decalcified, paraffin embedded femur. Any tips?? Thanks, Brett M. Connolly, Ph.D. Molecular Imaging Team Leader Merck Co., Inc. PO Box 4, WP-44K West Point, PA 19486 tel. 215-652-2501 fax. 215-993-6803 brett_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ 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
