[Histonet] RE: IHC on cells on coverslips

From:Tony Henwood


"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
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


-----Original Message-----
From: C.M. van der Loos [mailto:c.m.vanderloos@amc.uva.nl]
Sent: Thursday, 11 December 2003 6:52 PM
To: Histonet@pathology.swmed.edu
Cc: pruegg@colobio.com; anthonyh@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@colobio.com]
Sent: Wednesday, 10 December 2003 9:12 AM
To: Histonet@Pathology. Swmed. Edu
Cc: Ihcrg@Yahoogroups. Com
Subject: [IHCRG] IHC on cells on coverslips

Please advise how to manage IHC staining for cells grown on coverslips.

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