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
> 
> _________________________________________________________________
> MSN 8 helps eliminate e-mail viruses. Get 2 months FREE*.  
> http://join.msn.com/?page=features/virus
> 
> 


~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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."