Re: Lack of penetration of immunolabelling

Posted by RICHARD BURRY on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-Photobleaching-vs-Sensitized-Emission-FRET-results-tp4828946p4933715.html

Louis

 

One problem could be rinses after antibody incubations.  I have recently did experiments published in my Springer book” Immunocytochemistry – A Practical Guide for Biomedical Research”, looking at labeling after different numbers of rinses.  A low number of rinses after a primary antibody incubation greatly reduced labeling with no background.  The primary antibody on the sections was reacting with the secondary antibody in solution and preventing its penetration into the tissue.  The effect was that the concentration of the unbound secondary antibody was much lower than that added.  I found that 7 rinses were needed for the conditions I used to gain maximum labeling with the secondary antibody.  However, it is the lack of rinses after secondary antibody that increases background.

 

Richard Burry

----- Original Message -----
From: [hidden email]
Date: Tuesday, April 20, 2010 4:50 pm
Subject: Lack of penetration of immunolabelling
To: [hidden email]


> 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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Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814  Cell 614.638.3345  Fax [phone]