[Histonet] processing adipose tissue



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

tel : (33)561004179  fax :(33)561004001

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