[Histonet] Regarding a problem in manual paraffin embedding

[Massimo]

Hi,



I think that your problem is not an enough infiltration time into Xylene but It is a not complete 
dehydration of the specimen quite thick.
When it happens I come back to new absolute ethanol and then again into Xylene up to complete
clearification.
I process small pieces so my scheduled times in the procedure are not suitable for your thick specimen.
It is not advisable a long time in Xylene because it makes the piece fragile on cutting operation.
It is better to process them for a longer time into 95° and/or 100° ethanol.
Preferably in 95° ethanol because the 100° one makes the pieces too hard.

I use an intermediate mix of 50% 95° and 50% 100° ethanol solution too.
It is advisable to make two or more steps in new ethanol and Xylene solutions.

Kind Regards and wishing for a Happy New Year

Massimo Tosi
"A humble Chemical Engineer who loves Histology"

________________________________
 Da: Chirayu Sharma <chirayu2011 <@t> gmail.com>
A: histonet <@t> lists.utsouthwestern.edu 
Inviato: Martedì 1 Gennaio 2013 23:17
Oggetto: [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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