RE: cell culture for IHC

From:Chris van der Loos

Hi Laura,
In the past I did some IHC on cell cultures grown on plastic disposables. 
This created an extra problem with respect to the fixation since 
acetone-fixation was not possible for obvious reasons. Finally, we used a 
4% paraformaldehyde (freshly prepared in PBS) for 5 min at RT. This short 
aldehyde-fixation does not cause any cross-linking and therefore antigen 
retrieval is not needed (impossible too, because of the plastics!)
During ALL IHC incubation steps and washings (from endo PO blocking up to 
chromogen) we included 0.1% saponin to open up the cell membranes (to get 
your IHC reagents in and out of the cells).
Lots of success!

Chris van der Loos
Dept. of Cardiovascular Pathology
Academic Medical Center
Amsterdam, Netherlands

Laura wrote:
 >Date: 13 Feb 2003 10:30:26 -0600
 >Subject: Cell Culture for IHC
 >I have been asked to do an immunohistochemistry stain (West Nile Virus) 
on cell
 >culture. I usually on worked with paraffin blocks and smears. I'm not even
 >sure of the questions to ask, but here it goes....
 >In growing the cells, can I used glass slides (I don't have any plastic)?
 >Are the glass slides plain or charged?
 >How do I fix the cells? (I know if they're not fixed in formalin there will
 >be no antigen retrieval.)
 >Anything special I need to know before the jump such as potential 
problems or
 >background staining?
 >Thanks in advance,
 >Laura Bliven
 >Marshfield Laboratories

Kathleen wrote:
 >Date: 13 Feb 2003 13:01:22 -0600
 >From: Kathleen Spencer 
 >Subject: Re: Cell Culture for IHC
 >In the past I did IHC on cultured hippocampal neurons grown on round
 >glass coverslips in tiny petri dishes. The coverslips were coated with
 >poly-l-lysine after soaking in nitric acid. Everything was done with
 >sterile technique of course. The fixation of the cells and the staining
 >took place in the petri dish, suctioning off each solution. The
 >coverslips were placed cell side down on a slide with vectashield for
 >Hope this helps,

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