[Histonet] Regarding a problem in manual paraffin embedding

[Rittman, Barry R]

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-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Chirayu Sharma [chirayu2011 <@t> gmail.com]
Sent: Tuesday, January 01, 2013 4:17 PM
To: histonet <@t> 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
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