[Histonet] Regarding a problem in manual paraffin embedding

[Rene J Buesa]

Chirayu:
I think there are several issues in your protocol:
1- the fixation time looks well but when you wash the fixed specimen overnight with running water you "unfix" the tissue because formalin fixation is totally reversible. You end with an unfixed tissue. After fixation do not wash the fixed tissues and start the dehydration directly.
2- the graduated dehydration looks well but you should double the dehydration steps times.
3- you complain that the heads do not "clear" and they will never clear. It is a common misconception that xylene is a "clearant" and it is not. Xylene is a paraffin wax solvent. The term "clearant" is misused in this case and refers to some chemicals, like aniline oil and especially cedar-wood oil that have the ability to make dehydrated tissues transparent but are no good paraffin solvents and that is why after cedar-wood oil some paraffin solvent, like benzene and xylene, has to be used. If a tissue was made transparent by the aniline oil or the cedar-wood oil, it remained transparent after being immersed in benzene or xylene. Xylene by itself will NOT make tissues transparent. So do not worry about that, what you need is to increase the time in xylene using at least 3 changes of 3 hours each.
4- the description of the sections separating in contact with the water is a sign of incomplete infiltration (too little time in xylene and probably in paraffin also) as is the whitish appearance of tissue when sectioning.
Summing up, you should eliminate the water washing, duplicate the dehydration times and have 3 xylene changes of 3 hours each (=9 hours).
Finally you do not wash the tissue in paraffin; the tissues have to remain in the paraffin. Try using 4 paraffin infiltration periods of at least 2 hours each.
René J.

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
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