Posted by
Tamara Howard on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-Photobleaching-vs-Sensitized-Emission-FRET-results-tp4828946p4934217.html
How were the samples fixed? Do you fix the sample & then
freeze, or are these fresh-frozen and then the sections
fixed? Are you certain that your problem antibodies are
compatible with the fixation?
Tamara
On Tue, 20 Apr 2010 16:45:37 -0400
[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]
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Tamara Howard
Cell Biology & Physiology
UNM-HSC
Albuquerque, NM
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