[Histonet] Manual fixation/processing of reconstructed skincultures grown on polycarbonate filters

[Elizabeth Chlipala]

Colin

We have worked quite a bit with the epioral and epigingival cultures
from Mattek.  We have stained with H&E, Ki-67 and Cleaved caspase 3. We
processed these specimens on a tissue processor, but you will be able to
do this by hand.  I'm not sure of the size of your culture wells, but we
used an 8mm biopsy punch to remove the constructs from the wells,
processed the construct whole between two biopsy pads and bisected with
a razor blade prior to embedding on end.  As far as fixation 10% NBF
will work.  The wells were labeled on the side and the entire well was
placed in a 50 ml conical tube with fixative.  After adequate fixation
the specimens were grossed, processed and embedded into paraffin.  We
processed during the day (not overnight) at 15 minutes per station.  We
sectioned at 4 microns and stained with both H&E and various
Immunohistochemical stains, I can send you images if you are interested.
If you need more info, give me a call or e-mail me back.  Also we are
writing an article that will be in the spring edition of Histologic that
basically covers the methods (both processing and analysis) we used to
assess proliferation in these in vitro tissue models.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
P.O. Box 18592
Boulder, Colorado 80308
Office: (303) 735-5001
Fax: (303) 735-3540
liz <@t> premierlab.com
www.premierlab.com
 
Ship to Address:
Premier Laboratory
University of Colorado
MCDB, Room A3B40
Boulder, Colorado 80309

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Colin
Smith
Sent: Wednesday, January 26, 2005 8:07 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Manual fixation/processing of reconstructed
skincultures grown on polycarbonate filters

Can anyone help?
I am currently setting up a basic histology system for analysing
reconstructed tissues grown in culture on polycarbonate filters, similar
to
Mattek's Epiderm. We currently have begged borrowed or stolen,
equipment-wise:
two water baths (ambient - 100C), a circulating warm air oven, a rocking
microtome and cold blocks, along with glass staining jars/racks, forceps
etc.
At the moment, equipment such as a wax dispenser, purpose built cooling
plates etc are out of the question until I can show that we can show
that the
model we are growing is validated, which will include
immunohistochemistry of
cytokeratin and cell adhesion markers etc. Thus we are in the catch 22
situation of having to prove something without the equipment needed to
do the
job, in order to justify buying the equipment!! But thats accountants
for
you, go figure.
Anyway, can anyone give any useful pointers/protocols for
fixing/processing/embedding/sectioning such tissues using such primative
equipment
Any help, no matter how trivial will be greatfully received.
 
Many Thanks in Advance,
 
Colin Smith BSc CBiol MIBiol Eurotox Registered Toxicologist
In Vitro Toxicologist 
Thor Specialities UK Ltd
Wincham Avenue,
Northwich
CW9 6GB
 
Tel: +44 1606 818873
Fax: +44 1606 818801
colins <@t> thor.uk.com
 
 

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