RE: cell culture for IHC

From:"Georger, Mary"

The chambered slides we use are from Nalge Nunc, and are called  Lab-Tek II
Chamber Slide System. We use the 4 chamber slide, with the "removable
chambers" ,  Nalge Nunc catalogue #154526. We buy them through VWR. They are
also available with 1,2 or 8 chambers.

Mary Georger
Center for Cardiovascular Research
University of Rochester Medical Center
KMRB Room 2-9816
601 Elmwood Avenue.
PO Box 679
Rochester, New York  14642

> ----------
> From: 	Luis Chiriboga
> Sent: 	Friday, February 14, 2003 7:16 AM
> To: 	Chris van der Loos;
> Cc:
> Subject: 	RE: cell culture for IHC
> Hi All
> I have seen a system that allows you to grow cells directly on glass
> slides
> .  I do not know the name of  the system since I have never actually used
> for cell culture but do have a collaborator who use it exclusively.
> Essentially,  It is a clear plastic box without bottom that fits over the
> glass slide (excluding the label area).  A "rubber?" contact adhesive is
> used(do not know name or composition) to attach the box to the slide
> surface
> such that a water tight seal is made. The box comes with a lid and the
> cells
> can be grown directly on the glass slide. The benefits of this system are
> that any fixative can be used.  Also,  if you are doing manual staining,
> the adhesive acts as a hydrophobic barrier so you do not have to use a PAP
> pen.  The main drawback is if your are doing automated staining.  You have
> to scrape the adhesive off the slide for cap gap instruments or ventana
> instruments because it will impeded the movement/mixing of solutions.  In
> addition,  the entire slide is covered with cells so it would require a
> lot
> of reagent when doing manual staining, we have gone around this by wiping
> of
> areas of the slide Ill see if I can come up with a name.
> Thought this might be another option (although the lack of vendor
> information  makes it this post quite useless :-)
> Regards
> Luis
> -----Original Message-----
> From: Chris van der Loos []
> Sent: Friday, February 14, 2003 2:53 AM
> To:
> Cc:
> Subject: RE: cell culture for IHC
> 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
>  >From:
>  >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
>  >microsopy.
>  >Hope this helps,
>  >Kathleen

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