Re: Problems with immuno on frozen tissue

From:Amos Brooks <>

    Are you air drying the frozen sections before or after you acetone fix
them? From your message I gathered that your poor morphology could be lack
of fixation. We immediatly after a section is cut place the slide in acetone
to fix, then we air dry them.
    Alternatively you could use a 10% neutral buffered formalin fixation
step followed by heat induced antigen retrieval, but depending on what you
are trying to do this might seem counter productive. (Why crosslink your
proteins just to uncrosslink them right after right?) Some times you'd be
surprised at the circuitous logic of  immunohistochemistry.
    Another possible source of staining problems could arise from the
peroxidase blocking you are using. Try a 3% solution of hydrogen peroxide in
methanol or distilled water, instead of in TBS. The methanol usually helps
quench the peroxidase.
good luck
Amos Brooks

----- Original Message -----
From: <>
To: <>
Sent: Thursday, April 26, 2001 5:43 PM
Subject: Re: Problems with immuno on frozen tissue

> Good afternoon fellow histonetters,
>                                                     Our lab has been
having a
> tough time staining a frozen prostate with p53 antibody.  The stats are as
> follows:  Sections cut at 4 microns on a cryostat, sections let air dry
> 2-3 hours, fixed in cold acetone for 10 minutes, let air dry and then put
in TBS
> buffer pH 8.0 for 10 minutes.  Sections then blocked for endogenous
> with 250 microliters of 30% H2O2 in 50 mls of1X PBS for 10 minutes and
> rinsed.  Blocked for avidin and biotin followed by incubation with Dako Ab
> p53 clone DO-7 for 1 hour at room temp(used a Tris-Hcl Ab buffer),
followed by a
> 15 minute incubation with Dako LSAB+AP link, then Dako LSAB+AP strepavidin
> developed with Fast Red chromagen for 15 minutes(reagent made right before
> was used).  When we look at the slides we have no positive cells(we were
> there were some).  Not oinly do we have no + cells, the morphology of the
> is extremely messy.  It almost looks as if alkl the cells have lost their
> membranes and burst open.  I expected the morphology to be bad due to the
> acetone fixation but npot to the degree that I have seen.  Is this why
none of
> the slides are working?
> Any help you can offer is greatly appreciated.  The tissue block has been
> mounted on a chuck and stored at -80 C wrapped in aluminum foil and is
being cut
> by a very experienced histotech.
> Sincerely,
> Christine Klein
> MD Anderson

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