Chirayu, I am not surprise you have this problem, it's a classic issue for many lab (if they don't do research). Any time if Mouse embryo are older than Day 13 (E13), you need a small sharp scissor to slit open the mouse neck/skull & abdominal (> E13) in order to have properly fixation. It got nothing to do with tissue processing. You can process overnight (1hr - 1.5 hr per steps). Good Luck! madelein...@elcaminohospital.org
On Wed, Jan 2, 2013 at 10:00 AM, <histonet-requ...@lists.utsouthwestern.edu> wrote: > From: Chirayu Sharma <chirayu2...@gmail.com> > To: histonet@lists.utsouthwestern.edu > Sent: Tuesday, January 1, 2013 5:17 PM > 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