[Histonet] Processing Fat for Paraffin

[Laurie Reilly]

Dear Paula,

Don't knock the Autotechnicon, they can still do a great job of processing.
We processed with a Shandon-Elliott Duplex up until 3 years ago and it was
great (apart from the fumes in the lab).

We run the cycle below as a 16hour, overnight process successfully.

I don't know if your citrisolv will be as effective at dissolving fat as
xylene, but it would be easy to do a little trial.

The major problem with processing fatty tissues, assuming that they are
fixed properly, is that Ethanol is not a good solvent for fat and therefore
connot penetrate the tissues completely, so the tissues are inadequately
dehydrated.
We have had some success with lipomas by adding a "degreasing" step of
xylene into the processing schedule.
70% ethanol
80% ethanol
90% ethanol
95% ethanol
Absolute ethanol
Xylene
Absolute ethanol
Xylene
Xylene
Paraffin
Paraffin
Paraffin
The first Absolute ethanol will dehydrate the tissue to some extent. The
next Xylene step will remove most of the fat and then the second Absolute
ethanol can complete the dehydration.

A compromise situation that we use routinely is to have
 Absolute ethanol 
50:50 Absolute ethanol:Xylene 
Xylene 
This is not quite as effective but it is less disruptive to the normal
schedule and handles moderately fatty tissues.

Good luck with your fat.

Regards,    Laurie.


Mr. Laurie REILLY
Histopathology
School of  Veterinary and Biomedical Sciences
James Cook University
Townsville  Qld.  4811
Australia.

Phone 07 4781 4468

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Va Paula
Sicurello
Sent: Friday, 1 May 2009 8:22 AM
To: HistoNet; MSA BB
Subject: [Histonet] Processing Fat for Paraffin


Hello out there in fat processing land,

I have been given human fat samples and need to embed them in paraffin.  In
the past I've used a VIP processor for this and now I have an Autotechnicon
(vintage dual model) with a timing wheel.

I know I need to process these fatties slowly, my question is--can I use 2
hours per step and have it turn out OK?  I have a timing wheel punched out
for 2 hour steps.

My steps would be alcohols: 70, 80, 95 x 2, 100 x 3, Citrisolv x3, paraffin
x2 and another paraffin step under vacuum.

Let me know your wise and experienced opinions or protocols.  I don't have
anything else to use except the 43 year old Autotechnicon so don't even
suggest it.  You make me feel bad that I can't get my research foundation to
buy me something new or even newer.  ;-)

Stewing in somebody else's fat (eew!),

Paula  :-)

 
Paula Sicurello
VA Medical Center San Diego
Veterans Medical Research Foundation (VMRF) 
Core Research Imaging Center
3350 La Jolla Village Dr., MC151
San Diego, CA 92161
858-552-8585 x2397


      


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