[Histonet] Réf. : RE: [Histonet] processing adipose tissue

[Nancy.Walker <@t> sanofi-synthelabo.com]

Hello,
I'm not really interested in the lipids but rather the proteins and mRNA of
adipocytes and/or stromal tissue. The most basic question is whether the
target gene is expressed in the stoma or by the adipocyte. If I'm using in
situ hybridisation the mRNA is found in the cytoplasm not in the lipid
droplet, which indeed will be removed by the paraffination et
deparafination. I've been told that  frozen tissue often needs to be
delipidized to render the mRNA more accessible to hybridization.  In the
case of IHC the paraffin tissue also has the same advantage (better
accessibility, better cellular morphology - particulary if one is trying to
differentiate mulitlocular adipocytes, histiocytes, blood vessels). Only
for proteins secreted by the adipocyte and stored in the vacuole would
paraffin slices be not the best bet. However even in some cases of secreted
proteins like ACRP, some protein can be detected on the cytoplasmic rim of
a adipocyte even after parrafination.

correct me if I'm wrong or imprecise,

yours truely,
Nancy Walker
Molecular Biology Scientist

Sanofi-Synthelbo Research
B.P. 37 Labége Innopole
31676 LABEGE CEDEX FRANCE

nancy.walker <@t> sanofi-synthelabo.com
tel : (33)561004179  fax :(33)561004001




                                                                                                                      
                      "George Cole"                                                                                   
                      <georgecole <@t> ev1.         Pour :   Nancy Walker/FR-LABEGE/RESEARCH/SANOFI <@t> Research               
                      net>                     cc :                                                                   
                                               Objet :  RE: [Histonet] processing adipose tissue                      
                      21/11/03 12:31                                                                                  
                                                                                                                      
                                                                                                                      




Nancy,
No advice here, but questions:---doesn't the clearing process in
paraffin impregnation interfere with the nature of adipose tissue?  It
can't really tell you anything about that which was melted away in the
clearing step. Are frozen sections useful for your work? Can you not get
sections that are useful to you with lipids cut in a good cryostat at
-25 to -30C on a very sharp knife?
georgecole <@t> ev1.net

-----Original Message-----
From: histonet-admin <@t> lists.utsouthwestern.edu
[mailto:histonet-admin <@t> lists.utsouthwestern.edu] On Behalf Of
Nancy.Walker <@t> sanofi-synthelabo.com
Sent: Friday, November 21, 2003 1:25 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] processing adipose tissue

Bonjour,

I have adipose tissue to process (fix and paraffin embedding) for in
situ
hybridization (ISH) and IHC experiments. I've done this in the past with
a
program and got pretty good tissue morphology and done some experiments,
but since I was told by Gayle Callis that my processing schedules for
rodent brain and embryo are probably excessively long.  In fact in a
recent
experiment where I included an embryo slide prepared by Novagen; Signals
were much better with the commercially supplied slide.

So does anyone have any advice on processing fat?  I'm using small
samples
of epididymal  fat (0.5g - 1 g per sample ) that have been fixed
overnight
in PFA 4% and washed in PBS for 6h.
This is the program we used :
     alcohol     70%         1h
            95%         2X1h
            100%        2X1h
            100%        2h
           clearify          3X1h
           paraffine         3X1h30  (Sakura tissue tech III)

     (Our embedding machine does have possibilities to do vacuum and
heat).
Before  doing  IHC  or  ISH  we  just deparaffin with xylene     (2x5')
and
rehydrate.  We do Protein K before in situ and microwave or trypsin or
EDTA
before  IHC  .  Are there some specific           pretreatments for
adipose
tissue for delipidizing that might improve probe or antibody
accessibility.

     I appreciate your advice and thanks for your valuable time,
     Nancy Walker
Molecular Biology Scientist

Sanofi-Synthelbo Research
B.P. 37 Labége Innopole
31676 LABEGE CEDEX FRANCE

nancy.walker <@t> sanofi-synthelabo.com
tel : (33)561004179  fax :(33)561004001





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