Re: Problems with immuno on frozen tissue -Reply

From:Tony Henwood <>

Dear Christine,
A few questions and suggestions:
Why block endogenous peroxidase when you are using alkaline
phosphatase as your label?
Try immediate fixation in 95% ethanol, followed by rinsing in buffer and
then progressing with your immunohtochemical procedure. Don't let the
sections dry out.

Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318 

>>> <> 27/April/2001 07:43am >>>

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
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 for
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 and
developed with Fast Red chromagen for 15 minutes(reagent made right
before it
was used).  When we look at the slides we have no positive cells(we
were assured
there were some).  Not oinly do we have no + cells, the morphology of
the cells
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
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
mounted on a chuck and stored at -80 C wrapped in aluminum foil and is
being cut
by a very experienced histotech.

Christine Klein
MD Anderson

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