immunofluorescence background

Richard R. Hardy hardy at mighty.fccc.edu
Fri Mar 17 14:32:44 EST 1995


In article <01HO8F46MB8Y8WXWK7 at VAX.CS.HSCSYR.EDU>,
gilbertd at VAX.CS.HSCSYR.EDU (Dave Gilbert) wrote:

> Does anyone have any tricks to reduce apparently non-specific association
> of polyclonal antibodies.  We have tried blocking steps with serum,
> however, our immunofluorescent signals with antibody are still only
> marginally above signals obtained with pre-immune serum.
> 
> Thanks.
> 
> Dave Gilbert
> Department of Biochemistry and Molecular Biology
> SUNY Health Science Center
> 750 E. Adams St.
> Syracuse, N.Y. 13210
> Tel:(315) 464-8723
> FAX:(315) 464-8750

Not much of a "trick", but we always deaggregate the antibodies (labeled
antibodies often aggregate) immediately prior to dilution and staining. 
In a pinch, use 30 min at highest speed on a microfuge; better yet, use a
tabletop ultra (like an "AirFuge").  Some combinations are particularly
bad: rabbit IgG sticks very well to mouse lymphocytes; goat Ig is
preferred in our experience (difference in Fc binding?).  Good luck!

-- 
R. Hardy
Member, Institute for Cancer Research,
Fox Chase Cancer Center, Philadelphia, PA
(215) 728-2463



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