Hey Mike!
        Good to hear from you! How are they treating you down South? Miss you!
        Thanks for the suggestions. I'm a little concerned about taking out all
the calcium, because the cadherins in the cell junctions are
calcium-dependent. Most protocols actually increase the calcium in the fix
buffer. I've just left it with what is normal for Dulbecco's PBS with Ca
and Mg. I'm wondering about osmolarity, though, and you have some good
suggestions. I'll keep them in mind!
        Kathy




At 02:53 PM 11/26/01 -0600, you wrote:
>Hi Kathy!
>
>  Don't know if this'll help, but... since we're working on striated
>muscle, we make sure the fix has zero calcium, high EGTA (10 mM), and high
>magnesium (10 mM). The rationale is to prevent contractions via calcium
>influx or triggering store release while the fixative forms cross-links. We
>also add the fix very slowly. If this seems to help but doesn't completely
>solve the problem, you might even try a short pre-application of EGTA- or
>BAPTA-AM to buffer intracellular calcium (I've never tried this and you'd
>want independent verification that this doesn't affect the
>morphology/junctions you're interested in). You could also try cooling down
>the cultures and the fix before application. Best of luck, and hope to see
>you at ASCB.
>
>Mike
>
>~~~~~~~~~~~~~~~~~~~~~~~~~~~
>Michael B. Ferrari
>Assistant Professor
>Department of Biological Sciences
>University of Arkansas
>Fayetteville, Arkansas 72701
>Ph: 501-575-6372
>Lab/Fax: 501-575-5349
>http://biology.uark.edu/ferrari
>~~~~~~~~~~~~~~~~~~~~~~~~~~~
>
>
Kathy Spencer
The Scripps Research Institute
10550 N. Torrey Pines Road
IMM 24
La Jolla, CA 92037
(858) 784-9372
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