You wrote: 

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.

Why not make the fixative up that you are using, and take out the acetic
acid. You may have to adjust for its 5% volume with buffer? or water?
There is a commercial alcoholic formalin (Anatech) but not sure of alcohol
concentration, they have a website. There are recipes for alcoholic
formalin in other histotechnology books.  

The acetic acid in your fixative is damaging the DNA, causing poor
hematoxylin staining i.e. Acid hydrolysis of DNA/protein.  The alcohol
concentration is high, probably causing the shrinkage.  Maybe you need to
test different concentrations of alcohol in the fixative at the same
temperture and/or less time rather than more.  Just some thoughts.  

Acetic acid excessively swells cells, alcohol excessively shrinks cells and
that is why these are combined to offset the effects of each. 

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu