RE: cell block fixation

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From:Cynthia Favara <cfavara@atlas.niaid.nih.gov> (by way of histonet)
To:histonet <histonet@magicnet.net>
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Marcia,
	I don't have a protocol but am starting a project that requires
using cells from a suspension as positive and negative controls. Having
never done this before I would like some thoughts.  We are going to use
and NBF fix and an ETOH fix because we know the IHC works in the Elisa
assay under those conditions. Here is the proposed protocol.

	Spin cells 1000 rpm for 10 minutes
	Remove supernatant
	Add 10 ml of fixative [NBF or ETOH] Time??? Should only take an
hour or so??
	remove supernatant
	wash 2X in DPBS
	add 1.0% gelatin to pellet - just enough to get a semi solid
block to process
	wrap in tissue paper and process.

If you or anyone else has some other ideas I would be happy to hear.

Took a cours at SLC that suggested a cell suspension on a collagne
matrix. sopund lovely but since I am unable to get the product by Tues I
just need to wing it.
Thanks


Cynthia Favara
Rocky Mountain Laboratories
903 S 4th Street
Hamilton, MT 59840
ph: 406-363-9317
FAX: 406-363-9286
e-mail: cfavara@nih.gov

> ----------
> From: 	Marcia Bentz[SMTP:mb7x@virginia.edu]
> Sent: 	Thursday, October 08, 1998 11:04 AM
> To: 	Cathy Fragiskatos; histonet@pathology.swmed.edu
> Subject: 	Re: cell block fixation
>
> At 10:35 AM 10/8/98 +0000, Cathy Fragiskatos wrote:
> >
> >    Dear histonetters,
> >
> >    I was wondering if anyone out there has run a study on various
> >fixation protocols for cell blocks.  If so, I have a few questions
> for
> >these individuals:
> >
> >1-  What was the best overall fixation protocol for cell morphology?
> my guess would be 10% neutral buffered formalin and paraffin embedding
>
> >2-  What was the best fixation protocol for CD markers? (particularly
>
> >for B and T cell markers)
>
> for this it's best to have frozen sections fixed with acetone @ -20
> for at
> least a couple of minutes (some have told me 10 min, some have said as
> little as 30 sec) If you must do paraffins and want to use CD3 use the
> Dako
> Ab.
> >
> >    Any information forwarded would be greatly appreciated.
> >
> >    Best Regards,
> >
> >    Cathy Fragiskatos
> >    IHC Laboratory
> >    Mc Gill University/ Royal Victoria Hospital
> >    Montreal, Quebec.
> >
> >    e-mail: fragiska@pathology.lan.mcgill.ca
> >
> >
> >
> >
> Marcia Bentz
> Lab Specialist Senior
> UVA
> Dept Internal Medicine/GI
> 804-243-6895
>
>
>
>




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