[Histonet] Neuronal cell culture blebbing

[Jo Dee Fish]

Hello everyone,
 
I have a question about neuronal cell cultures, and although I know we are a
group who work primarily with tissues, sometimes these types of questions
come to my lab and I am intrigued enough to try to find an answer.  And I
though I would consult with my fellow histonetters to see if any of you have
any suggestions.
 
An investigator who works with neuronal cell cultures has fixed them with
3.5% paraformaldehyde in 1xPBS solution with success, twice.  Then the third
time he saw blebbing on the cell membranes, and after consulting with our
confocal microscopy expert, he changed to 4% paraformaldehyde (in, I
believe, the same buffer) and had the same results.  It turns out our expert
has had the same problem with seemingly healthy cells that develop the
blebbing upon fixation.  My first instinct was that he needs to adjust the
osmolarity of the solutions.  Then I fell back to my electron microscopy
experience and thought he may need to use a different fixative/buffering
system, such as glutaraldehyde in cacodylate buffer.  Does anyone here have
a good protocol for fixing  neurons in culture so that we can look at them
as close to their natural state as possible.  Seems like a simple
question...
 
Thanks!
Jo Dee
 
Jo Dee Fish

Research Technologist III

Gladstone Institute of Cardiovascular Disease 

Telephone: (415) 734-2567

Fax: (415) 355-0230

E-mail: jfish <@t> gladstone.ucsf.edu

Mailing address:  

The J. David Gladstone Institutes

1650 Owens Street

San Francisco, CA 94158