Posted by
Martin Wessendorf-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-Photobleaching-vs-Sensitized-Emission-FRET-results-tp4828946p4933674.html
Dear Louis--
It might simply be that the antibodies don't penetrate well--some don't.
In my experience, cryostat sections are more problematic than sections
that are stained free-floating and mounted on slides after the staining.
However, some antibodies appear just not to diffuse through tissue
easily.
Good luck!
Martin Wessendorf
[hidden email] wrote:
>
> Bonjour à tous,
>
> We are trying to (indirect) immunostain with antiboby on heart
> cryosections (14um). The proteins that we want to identified are from
> the cytoskeleton like desmin, myosin light chain, myosin heavy chain.
> We have a problem that all the staining stay on top of the tissue for
> those antibodies. Counterstaining for actin (phalloidin alexa
> conjugated) is pretty good through the thickness of the tissue. We
> permealized the tissue with Triton 0.5% , in the blocking solution
> (serum from the host of the 2nd Ab), for 1 hour and we incubate the
> primary overnight at 4C (with Triton 0.2% in the antibody diluent).
> Other antibodies against cytoskeleton protein (myosin light chain 7)
> are well stained over the thickness of the tissue using the same
> protocol and the same batch of tissues.
>
> Any clue that might help us?
>
> Louissssssss
> Louis Villeneuve
> Research Associate- Confocal Microscopy
> Heart Montreal Institute- Research Center
> 5000 East Belanger
> Montreal (Qc), Canada
> H1T 1C8
>
> 514-376-3330 ext 3511
> 514-376-1355 (Fax)
>
>
[hidden email]
--
Martin Wessendorf, Ph.D. office: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail:
[hidden email]