Dear Christine

If you are using alk phos, there is no need to block peroxidase as Tony has
mentioned. The peroxide and methanol block is what is damageing you slides.
While it is goog for paraffins it wrecks frozen sections. Try you protocol
without the peroxide block and hopefully you will see an improvement (or
I'll look stupid )

Good luck

John Auld


> -----Original Message-----
> From:	HistoNet Server [SMTP:histonet@pathology.swmed.edu]
> Sent:	Friday, April 27, 2001 6:09 AM
> To:	HistoNet Server
> Subject:	Daily Digest
> 
> 
> 
> 
> Here are the messages received yesterday!Date: 26 Apr 2001 22:55:41 -0500
> From: Tony Henwood <AnthonyH@chw.edu.au>
> Subject: Re: Problems with immuno on frozen tissue -Reply
> 
> 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 
> 
> >>> <cklein@mail.mdanderson.org> 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
> about
> 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
> peroxide
> with 250 microliters of 30% H2O2 in 50 mls of1X PBS for 10 minutes and
> then
> 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
> 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
>