[Histonet] RE: IHC on cells on coverslips

[C.M. van der Loos]

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