Chirayu a happy new year to you. I am guessing that there could be 3 possible problems. 1. It appears that the xylene is not changed - maybe the xylene should have more than one change to eliminate all the alcohol. 2. The alcohol you are using as 100% may have some traces of water in it. 3. There may be small traces of bone in the embryos and if so these will appear whitish and cause problems in cutting and so on. May need to eliminate this with a demineralizaton step before dehydration. Hope that these suggestion are helpful. Barry
From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Chirayu Sharma [chirayu2...@gmail.com] Sent: Tuesday, January 01, 2013 4:17 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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet