[Histonet] preparation of frozen sections

[gayle callis]

I can understand your question from the clinical point of view where you
want to cut the section, fix, stain and then examine for immediate
diagnosis. Not everyone does this. 

There are many of us, both in clinical and research, who do frozen sections
for other than diagnostic reasons.  We often need to do immunofluorescent or
enzyme immunohistochemical staining (chromogenic) for antigens e.g. CD4, CD8
and many others that will not withstand the kind of fixation you describe.
Often we need to do cold acetone fixation or some other solvent fixation for
this purpose.  

In that case, our histologic interpretation is based on a different handling
and fixation of a fresh tissue frozen section, and in our case, we cannot
just cut and immerse into alcohol.  

One, the fixative e.g. alcohol is not going to work for our antigen
Two, we perform cryomicrotomy on a piece of tissue, collecting as many as a
hundred sections, often serial and store the sections until staining
(immunostaining in particular) can be performed.   

Air drying is a form of fixation, and the act of picking up a section onto a
slide has been referred to as "flash drying".  The antigens we need to see
are better when air dried overnight, then fixed with either acetone or
acetone/alcohol (in the case, for murine CD markers).  Often air drying the
section at RT and then fixing with acetone, and storing these fixed sections
in a -80C freezer is very acceptable. 

If I were in a clinical setting and doing a routine H&E, I probably would do
exactly as you do now.  It is a matter of application. In our case,
immersion immediately into a fixative is not optimal for our
immunofluorescent or enzyme immunohistochemical results.  If we want to see
or identify where we are in a sample, we do exactly as you do, section and
immerse into fixative, and then do a rapid H&E stain when we can or within a
few minutes. We frequently immerse a frozen section into neutral buffered
formalin and fix later in the day, week or whenever to have excellent
morphology and staining results with an H&E.  

I hope this clarifies some parameters of performing cryotomy and staining
versus how you do it.  

Gayle M. Callis
HTL/HT/MTA(ASCP)
Bozeman MT  

 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tench, Bill
Sent: Tuesday, August 24, 2010 5:07 PM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] preparation of frozen sections

So as a pathologist, i have to ask you why you would want to air dry a
section?  From a diagnostic perspective, we consider air dried samples
unacceptable in my lab.  All of our standard histologic interpretation
is based on fixed sections.  So, why not drop the slide in a jar or ETOH
and keep it there until you are ready to stain?
 
Bill Tench
Associate Dir. Laboratory Services
Chief, Cytology Services
Palomar Medical Center
555 E. Valley Parkway
Escondido, California  92025
Bill.Tench <@t> pph.org
Voice: 760- 739-3037
Fax: 760-739-2604
 

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