Thanks to everybody for your detailed protocols. They will be very useful

Susanna

En/Na Mario Moronne ha escrit:

> 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