[Histonet] RE: IHC on cells on coverslips

[Tony Henwood]

Patti,

No I don't. 

HIER is used to reverse the effects of formalin fixation. Since I rarely use
formalin to fix my cytology smears (usually use 95% ethanol or Methacarn)I
do not use HIER on these smears.

Having said that, I suspect that there may be antigens out there that
require HIER and this is not due to the formalin fixation effect. I have not
come across these yet. The question arises how antibodies to these type of
antigens were raised. Were the antigens heated (similarly to HIER) prior to
immunisation?

Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html




-----Original Message-----
From: Patti Loykasek [mailto:ploykasek <@t> phenopath.com]
Sent: Tuesday, 16 December 2003 4:02 AM
To: Tony Henwood
Subject: Re: [Histonet] RE: IHC on cells on coverslips


Tony, I have a quick question for you. When you are doing IHC on ethanol
fixed smears - do you use any type of antigen retrieval? I would like to
optimize our IHC on smears, and am looking for some info. Thanks.

Patti Loykasek
Phenopath Laboratories
Seattle, WA

 
> Chris,
> 
> "Not really beneficial to many antigens"? I could list at least 50
> antibodies that work on ethanol fixed smears. Most of these are in routine
> use.
> eg: AE1AE3, Cam5.2, 5D3, Vimentin, Desmin, GFAP, PSAP, PSA, CEA, EMA,
> Skeletal muscle myosin, Actin,etc etc.
> 
> Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
> Laboratory Manager
> The Children's Hospital at  Westmead,
> Locked Bag 4001, Westmead, 2145, AUSTRALIA.
> Tel: 612 9845 3306
> Fax: 612 9845 3318
> 
> http://www.histosearch.com/homepages/TonyHenwood/default.html
> http://us.geocities.com/tonyhenwoodau/index.html
> 
> 
> -----Original Message-----
> From: C.M. van der Loos [mailto:c.m.vanderloos <@t> amc.uva.nl]
> Sent: Thursday, 11 December 2003 6:52 PM
> To: Histonet <@t> pathology.swmed.edu
> Cc: pruegg <@t> colobio.com; anthonyh <@t> chw.edu.au
> Subject: RE: IHC on cells on coverslips
> 
> 
> Dear Patsy,
> Please keep in mind that ethanol fixation as suggested by Tony Henwood, is
> very good for the cellular morphology, but not really beneficial to many
> epitopes/antigens! The acetone-fixation only works well for antigens at
the
> cell surface or structures that are at least tightly bound to anything. If
> you are dealing with an antigen that may leak out (cytokines, hormones,
grow
> factors, or any other small peptide) you need a 4% PFA fixation. Because
> cells grown on coverslips do have an intact outer membrane (and are not
> leaking!) the antigen of interest gets nicely fixed inside the cell. You
> need to add 0.1% saponin to all your reagents and buffers (from endogenous
> PO block up to chromogen step) to open up the cell membrane getting your
> reagents in and out.
> 
> Chris van der Loos
> Dept. of Cardiovascular Pathology
> Academical Medical Center
> Amsterdam - The Netherlands
> 
> -----Original Message-----
> From: Patsy Ruegg [mailto:pruegg <@t> colobio.com]
> Sent: Wednesday, 10 December 2003 9:12 AM
> To: Histonet <@t> Pathology. Swmed. Edu
> Cc: Ihcrg <@t> Yahoogroups. Com
> Subject: [IHCRG] IHC on cells on coverslips
> 
> Please advise how to manage IHC staining for cells grown on coverslips.
> Patsy
> 
> 
> 
> 
> 
> 
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