Lack of penetration of immunolabelling

Posted by Louis Villeneuve on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-Photobleaching-vs-Sensitized-Emission-FRET-results-tp4828946p4933609.html


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)

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