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Florian,

This is probably precipitated, coagulated, labeled secondary antibody.
Such precipitates are often present, even in newly purchased
secondaries. Try preparing your secondary antibody and then filter it
through a 0.45 um syringe filter before you use it. If you don't have
syringe filters, then centrifuge your secondary antibody at high G in a
clinical centrifuge for 5 minutes and then carefully remove the
supernatant without disturbing any precipitate that might be at the
bottom of the centrifuge tube and use the supernat for your
incubations. The syringe filters work best if you have them.

Hope that takes care of the problem,

Steve

____________________________________________________________________
Stephen C. Kempf
Associate Professor
Faculty Director - AU Hybridoma Facility
Department of Biological Sciences
131 Cary Hall
Auburn University, AL  36849

Tel: 334-844-3924
Fax: 334-844-4065

E-mail: [log in to unmask]
http://www.auburn.edu/academic/science_math/biology/faculty/kempf.htm
http://www.auburn.edu/research/hybridoma/
_____________________________________________________________________

On Wed, 10 Mar 2004, Florian Ulrich wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> --
> dear all,
>
> i am currently doing antibody stainings on paraffin sections.
> unfortunately, i am always getting very bright dots when i look at
> the staining under the confocal.when i do the control without first
> antibody, i also see these dots. therefore, they should clearly come
> from the 2nd antibody. it does not matter whether i use an alexa 488
> or alexa 546 coupled antibody, they both produce these effects. maybe
> the antibodies are bad, but they are from a commercial source and
> work fine in many applications. so, is this a probelm related to
> paraffin sectioning?
>
> i would appreciate any ideas or experience how to get around that
> problem and be forever grateful!
>
>
> best wishes,
>
> Florian
> ----------------------------------------------------------------------------------------------
> Florian Ulrich
> Heisenberg Lab
> Max-Planck-Institute for Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108
> 01307 Dresden
> Germany
> phone: (+49) 351 210 2689
> fax:    (+49) 351 210 1489
> email: [log in to unmask]
>