Immunostaining of nuclear proteins

Henk Veldman H.Veldman at lab.azu.nl
Fri Nov 5 03:53:19 EST 1999



Stephen Snowdy schreef:

> Does anyone know if methanol does a good job of permeabilizing adherent
> cells all the way down to the nucleus?  Are any special steps necessary
> to immunostain epitopes inside the nucleus?
> --
> Stephen Snowdy
> Department of Pharmacology
> College of Medicine
> University of North Carolina-Chapel Hill

Methanol for some 20 min should be sufficient. Acetone might be better, but
will probably dissolve the plastic of your culture dishes. In addition
Triton X-100 (0.2% in PBS as a preincubation or in all incubations) will in
some cases produce a more even and consistant staining; try it and see the
difference.
Remember, if there is any structure prone to unspecific binding of  primary
antibodies, it is the nucleus, and often this unspecific staining is very
difficult to get rid of.

Good luck.

Henk
--

H. Veldman
Laboratory for Experimental Neurology (NMZ)
Room G 02.320
University Medical Center Utrecht (AZU)
P.O. Box 85500
3508 GA  UTRECHT
The Netherlands
Tel: +31 30 2507565
Fax: +31 30 2542100
E-mail: H.Veldman at lab.azu.nl






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