[Histonet] antigen retrieval for 5 year pap smear slides

[Tony Henwood]

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: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of hao
zhang
Sent: Sunday, 14 September 2008 1:12 PM
To: histonet <@t> 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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