Hao,
It might be possible that not all of the mountant has been removed. Though I am
surprised that the staining is not better.
It just might be one of those instances where the keratin epitopes are damaged
during the 5 year storage.
Possibly the phosphotungstic acid, often included in the PAP stains, might also
be effecting the epitopes.
Again try these variables on buccal or sputum smears and compare the results.
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145
-----Original Message-----
From: hao zhang [mailto:[EMAIL PROTECTED]
Sent: Monday, 15 September 2008 12:22 PM
To: Tony Henwood
Subject: Re: [Histonet] antigen retrieval for 5 year pap smear slides
Hi, Tony, René and all,
I agree that pap smear is fixed by alcohol instead of formalin. Problem
for me now is that when I do IHC for pan-keratin stain I only find small part
cell stained (around 10%) even when I dont do antigen retrival by HIER. I know
it is around 5 year old slides but I dont know why I can not get higher
positive percentage because I used Hela cell as a control I got around 90%
positive percentage. In addition, I find a lot of pink color material remained
in the slide. I used 3% acid alcohol for destaining treatment and destaining
effect get improved but finally I still get less positive percentage for
pan-keratin. any suggest will be very appreciated.
On Sun, Sep 14, 2008 at 5:26 PM, Tony Henwood <[EMAIL PROTECTED]> wrote:
Do not bother to retrieve.
Usually PAP smears are not fixed in formalin but are alcohol
fixed.
Alcohol is an excellent fixative for intermediate filaments as
well as
keratins.
I would just de-coverslip the slides, rinse in buffer, block
endogenous
enzyme, add your diluted antibody (you will probably find that
you may
need to dilute your antibody further than for FFPE sections
(maybe up to
1-100 times more)) and finally demonstrate the bound antibody.
For titreing prepare some buccal smears, fix in the same
fixative used
for the PAP smears (95% ethanol or whatever commercial Cyto
spray fix
that is used), and work up the titreing as usual.
No antigen retrieval is required unless formalin cross-links
have been
formed.
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145
-----Original Message-----
From: [EMAIL PROTECTED]
[mailto:[EMAIL PROTECTED] On Behalf Of hao
zhang
Sent: Sunday, 14 September 2008 1:12 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] antigen retrieval for 5 year pap smear
slides
Hi, everyone
I want to do IHC from a 5 year old pap smear slides for
pan-keratin
stain.
I tried many methods of antigen retrieval protocols (10 mM
citric
buffer, ph 6; 1 mM EDTA, Ph8; 10 mM Tris buffer, Ph 10; ) and
heat at
95C for 40 minutes. I also used Triton x 100 (0.2%)
pretreatment 10
minutes and then SDS (0.5% or 0.25%) treatment for 10 minutes.
But all
method did not work. Any replies will be very appreciated.
--
Hao ZHANG, Ph.D
Scientist
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--
Hao ZHANG, Ph.D
Scientist
Diamics, Inc
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