Hi all (I should say- "Hi fixers"!)
Being a novice to various fixation techniques, I am really impressed that very
mild changes (few drops of 0.1% triton here and 0.01% glutaradehyde there) can
grossly affect the morphology of cells. Is there any published article or book
which discusses these subtle variations appropriate for different applications?
Any suggestions?
P.E. Hariharan
Max Delbruck Centre for Molecular Medicine,
FVK,
Wiltbergstr. 50,
Berlin 13125,
Germany
Guenter Giese wrote:
> Hi Kathy,
>
> check the osmolality of your culture medium, of prefixation and fixation
> medium. Mouse cells need a slightly higher osmolality compared to human
> cells, amd vice versa. I do not remember the exact numbers, but the
> difference I think is roughly 10 percent. This may account for shrinking /
> swelling effects.
>
> The inclusion of 50% glycerol in buffers added to non-permeabilized cells
> probably leads to severe osmotic effects (shrinking of cells, followed by
> re-swelling). Subsequent addition of buffer without glycerol (was this part
> of your protocol?) may lead to severe damage. Roughly 25 years ago, this
> was our method of choice for isolating nuclei by disruption of unfixed
> ciliate cells (Tetrahymena).
>
> Another issue is the use of glutaraldehyde. For some purposes, low
> concentrations (0.01 to 0.05%) in addition to 3.5 to 4% paraformaldehyde
> are advantageous.
>
> You may also try PEM (Pipes-EGTA-Mg-chloride)- buffer as a stabilizing
> buffer, and you may include detergent (e.g. 0.2% Triton X-100). A good
> reference regarding cytoskeletal preservation is: Vielkind and Swierenga,
> Histochemistry 91:81-88 (1989).
>
> By the way, did you check the pH of your PFA solution? Did you prepare it
> freshly (according to standard protocols) and did ***not*** use
> formaldehyde stabilized with 10 percent methanol? Did you take a freshly
> thawed aliquot of a frozen aliquot of 4% PFA? Sometimes slight variations
> in the protocol can make the difference.
>
> Hope this helps,
>
> Guenter
>
> At 11:13 26.11.2001 -0800, you wrote:
> >Hello All!
> > I have a question regarding fixation of tissue cultured cells.
> > When fixing
> >with 4% paraformaldehyde (10 min, room temp), the cells appear to shrink. I
> >realize this is a characteristic of this kind of fixation, but it really
> >messes up the morphology of cell-cell junctions. Methanol or acetone, or
> >the combination (-20, dry) doesn't seem to work either. I've tried
> >formaldehyde as well. I've also tried a cytoskeletal stabilizing buffer
> >(50% glycerol) that improved the morphology, but the cells still appear
> >stringy and pulled apart, instead of nicely abutting each other. We plate
> >our cells on a collagen/fibronectin matrix, which dramatically improved the
> >morphology, but it still lacks the pre-fixation appearance of confluent
> >cells. We have followed published protocols in papers about cell
> >junctions, but it just isn't working for us. Any suggestions?
> > Thanks!
> > Kathy
> >
> >
> >
> >
> >Kathy Spencer
> >The Scripps Research Institute
> >10550 N. Torrey Pines Road
> >IMM 24
> >La Jolla, CA 92037
> >(858) 784-9372
> >[log in to unmask]
>
> ------------------------------------------
> Dr. Guenter Giese
> Light Microscopy Facility
> Dept. of Biomedical Optics, MPI fuer Medizinische Forschung
> Jahnstr. 29, D-69120 Heidelberg, Germany
> Phone (+49) 6221-486-360 (Fax: -325)
> e-mail: [log in to unmask]
> http://sun0.mpimf-heidelberg.mpg.de/~ggiese/lightmicro/index.html
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