Mario,

Thanks for the additional tips for tubulin staining.

Susan Garfield

-----Original Message-----
From: Mario Moronne [mailto:[log in to unmask]]
Sent: Wednesday, March 07, 2001 1:51 PM
To: [log in to unmask]
Subject: Re: immunofluorescence MetOH GFP


Susanna,

I spent a considerable amount of time getting a reliable tubulin
labeling protocol. It is always a bit tricky but MeOH or cold acetone
treatment never gave labeling that remotely approached what I got
using a paraformaldehyde protocol. My method most closely matches
that of Susan Garfield (see her message). I do have a couple of
differences compared to the latter in that the first treatment I use
after rinsing with iso-osmotic microtubule stabilizing buffer (same
as Susan's but with enough additional PBS to keep cells from
shrinking) is treatment with a mild membrane permeant
homobifunctional succinimidyl ester (DSSD from Pierce) at about 0.2
mg/ml at 37 degrees. After a 10 min incubation, we next treat with
taxol (sorry I will have to check the concentration but I think about
50-100 ug/ml). Again this is all at 37 degrees to ensure that the
tubules do not depolymerize. After another several minutes, 4%
paraformaldehyde is added and incubated for an hour. The latter step
can be done at room temperature.

For microtubule labeling I was always happiest with the primary from
ICN (expensive but reliable) done in the standard way with 0.2%
Triton to help penetration and SuperBlock from Pierce or equivalent
for blocking. As for GFP, the DSSD step could be a problem and you
might omit it. But everything else, especially keeping the cells at
37 degrees until you get the paraformaldehyde in is important.

Good luck,

Mario


>Dear confocalist,
>
>I'm trying to make immunofluorescence of Tubulin in GFP transfected
>cells. I have a good staining fixing with cold methanol but I loose the
>GFP signal. With PF the tubulin staining is not so good. Any suggestion?
>
>
>Susanna
>

--
_____________________________________________________________________
Mario M. Moronne, Ph.D.
NanoMed Technologies
ph (510) 528-2400
FAX (510) 528-8076
Berkeley, CA
94706