Re: Fixative for cells grown on coverslip
Ioana,
Without alcohol (or methanol-this would have been my choice), your
fixative will freeze. But as long as the overall concentration of
alcohol in the final fixative is greater than around 35%, it should
keep the solution from freezing at that temperature. So, what if you
reduced the alcohol concentration (if possible-I have no way telling
what the final concentration is currently) to reduce the shrinkage
effects?
That's my amateurish two cents worth! I'll bet John Kiernan will have
a definitive answer for you when he gets around to replying. Good
luck with it.
Greg
Date sent: Fri, 23 May 2003 18:01:14 +0000
From: Ioana Croteau
Subject: Fixative for cells grown on coverslip
To: Histonet@pathology.swmed.edu
> Hello everyone,
>
> I would like some advice from the rich store of information in your
> collective experience! I work in research. My current project involves
> fixing chinese hamster fibroblast cells grown to a monolayer on coverslips.
> The fixing usually occurs at about -10C. My current fixative is based on a
> recipe from "Humason's Animal Tissue Techniques", 5th edition (1997) and is
> as follows:
> 10% aqueous formaldehyde 37-40%
> 5% glacial acetic acid
> 85% absolute ethanol
> The idea was to get particularly good nuclear fixation, since we are
> using a fluorescent nuclear stain (Syto 13, Molecular Probes) to look at
> fine detail of the nucleus. We are studying intracellular ice formation and
> the pattern it produces in the nucleus with Syto is characteristic. I am
> also staining with H&E, to see if the pattern is visible with this stain as
> well. I haven't had very good results so far; it seems the cells are
> shrinking excessively, not preserving the cellular architecture well. The
> Syto looks OK, but H&E is quite poor. I am fixing for abot 20 minutes at
> this temperature. I have tried increasing my fixation time up to1i hour,
> with no improvement. I'm thinking I would like to try another fixative,
> without so much alcohol, which I believe causes excessive tissue shrinking.
> Does anyone have any ideas, recipes or suggestions? Keep in mind the
> fixing is occurring at below 0C , which is very different from a routine
> pathology lab. Also, there is no sectioning or processing involved, so there
> are no issues with that.
> Thank you for your help,
>
> Ioana Croteau
> Research Assistant
> Canadian Blood Services
> Edmonton, Alberta, Canada
> (780)702-2534
> ioana_croteau@hotmail.com
>
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~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Charlottetown, P.E.I.
Canada, C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
"A farmer is a person outstanding in their field."
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