Re: Lack of penetration of immunolabelling

Posted by Phillips, Thomas E. on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-Photobleaching-vs-Sensitized-Emission-FRET-results-tp4828946p4934032.html

Another consideration is whether your primary is an IgM or IgG. IgM's are considerably bigger but a non-insignificant proportion of non-commercial primaries and even some commercial ones are IgM. Phalloidin's MW = 790 while an IgG is closer to 150,000 and an IgM 5x that.


Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Glen MacDonald
Sent: Tuesday, April 20, 2010 5:07 PM
To: [hidden email]
Subject: Re: Lack of penetration of immunolabelling

What is the length of incubation for the secondary antibody?  The typical 1-2 hr. incubation tends to be too short for most tissue sections.  


Regards,
Glen

On Apr 20, 2010, at 1:45 PM, [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]

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]