[Histonet] Re: Histonet Digest, Vol 110, Issue 2

[Madeleine Huey]

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!
madeleine_h <@t> elcaminohospital.org



On Wed, Jan 2, 2013 at 10:00 AM,
<histonet-request <@t> lists.utsouthwestern.edu> wrote:
> From: Chirayu Sharma <chirayu2011 <@t> gmail.com>
> To: histonet <@t> 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