Hi,

In particular, with nestin and some other structural proteins, fixation 
  may also be an issue. For a number of best available antibodies, PFA 
is not optimal, but acetone works well. If needed, please contact prof. 
Ismo Virtanen at ismo.virtanen at helsinki.fi for details.

Best regards Pertti



[log in to unmask] 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)
> 
> [log in to unmask]


-- 
Pertti Panula
Professor, Vice Dean of Research
Neuroscience Center
Institute of Biomedicine/Anatomy
Faculty of Medicine
POB 63, 00014 University of Helsinki
Finland
Phone: +358 9 19125263
Fax: +358 9 191 25261
Mobile: +358 40 5922 323
pertti.panula at helsinki.fi
http://www.helsinki.fi/neurosci/panula.htm