[Histonet] Regarding a problem in manual paraffin embedding

[Anatoli Gleiberman]

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: AGleiberman <@t> cbiolabs.com


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Chirayu Sharma
Sent: Tuesday, January 01, 2013 5:18 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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