Chirayu, Usual problems with heads of late mouse embryos is poor dehydrations. My recommendation is to use isopropanol instead of absolute ethanol.
My protocol that worked very well for many years was: Fixation with NBF for 4-6h, short rinse with PBS and storage at 70% ethanol overnight (Alternative fixation - 6 parts of absolute ethanol, 3 parts of distilled water, 1 part of 37% formaldehyde - 4-6 h, transfer in 70% ethanol overnight) 4 changes of isopropanol, 1h each 3 changes of xylene - 45 min each 3 changes of paraffin 1h each. Anatoli Gleiberman, PhD Director of Histopathology Cleveland Biolabs, Inc 73 High Street Buffalo, NY 14203 phone:716-849-6810 ext.354 fax:716-849-6817 e-mail: agleiber...@cbiolabs.com -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Chirayu Sharma Sent: Tuesday, January 01, 2013 5:18 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Regarding a problem in manual paraffin embedding Hello Histonetters I am having a problem in Manual Paraffin Embedding of Embryonic Day 16-18 mice heads. My protocol for Paraffin embedding is Fix in 4%NBF for 2 days followed by rinsing the whole head of mice in running water for overnight. Series of ethanol wash 50% -4 hrs 70% 4 hrs 96% 4 hrs 100% 4 hrs Xylene-5 hours Series of Paraffin wash each consisting of 30 minutes interval till the smell of xylene is not there in the Paraffin bath. Finally prepare the block. My problem is even after putting in Xylene for 5 hours I didn't see clearing of mice heads. I tried with one spare mice heads and kept in xylene for 8 hours but I did not see clearing too. Later I put the same head still without clearing in Parafin and prepare the block, I found the head to be soft and the initial part of the head facing the microtome is white. And flecks of wax could be removed easily from the top of tissue. Another problem is even these tissues are able to cut in some instances the sections tear when putting in water bath and they look something whitish appearance when placed in slides. My problem is I cannot remove the brain and skin for penetration of xylene as my study area does not allow me. It would be so nice if somebody would share me their Manual Paraffin Embedding protocol of whole head of mice in embryonic day 17-18 and Postnatal day 0 and 1 so that I can get rid of these problems. Chirayu _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This communication may contain privileged information. It is intended solely for the use of the addressee. If you are not the intended recipient, you are strictly prohibited from disclosing, copying, distributing or using any of this information. If you received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. This communication may contain nonpublic information about individuals and businesses subject to the restrictions of the Gramm-Leach-Bliley Act. You may not directly or indirectly reuse or redisclose such information for any purpose other than to provide the services for which you are receiving the information. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
