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We are also doing some research using myotubes but are having a hard time
keeping them adherent to the coverslips. Most of them end up falling off
after a couple washes with PBS. Have you encountered this problem? If so,
have you been able to correct for it?

-----Original Message-----
From: Brero Alessandro [mailto:[log in to unmask]]
Sent: Thursday, March 21, 2002 9:36 AM
To: [log in to unmask]
Subject: myotube fixation.



Hello confocalists,

sorry but I have still another fixation question. I am working on mouse
myoblast cells, which I differentiate into myotubes. I've read a post from
M. Ferrari on the confocal list, concerning the fixation of cells with zero
calcium, but as his maill adress seems to have changed, I post this on the
list. I'm interested in different aspects of chromatin distribution in
myotube nuclei, which I fix in 4% PFA/1xPBS and permeabilize in 0.5%
Triton/1xPBS. While the non differentiated myoblast nuclei look perfectly
smooth, the myotube nuclei look kind of distorted and twisted. So my first
thought was that maybe some kind of contraction is going on before/during
fixation, that leads to this presumably aberrant nuclear morphology. Hence I
would like to try a fixation method that prevents any of these movements. On
the post a few month ago M.Ferrari mentioned 10mM EGTA and 10mM Mg++. Does
anybody have experience in fixing myotubes, or protocols for zero calcium
fixations (considering osmolarity etc.), or at least M. Ferraris new email
adress??

Thanks for any suggestions.

Greetings from Munich,

Sandro.


Dipl. Biol. Alessandro Brero
Universität München
Department Biologie II Humangenetik
Richard-Wagnerstr.10/I
80333 München
Tel.: 004989/2180-6713
Fax:004989/21806719
E-mail: [log in to unmask] <mailto:[log in to unmask]>