Re: [Histonet] My frustrating experience with double fluorescence staining!

From:Adam Perry

Shouldn't your blocking buffer use donkey serum, since both of your secondaries are generated in donkey?  I would think that the rabbit serum block with your first secondary antibody (donkey anti-rabbit) would produce high background and non-specific staining...


Department of Physiology and Biophysics
University of Illinois at Chicago
Chicago, IL 60612
 Blocking Buffer (10% Rabbit serum; 5%
BSA; 0.1% Tween 20) for 1 hour at room temperature;

 Stain 1: Rabbit anti-mouse antibody, and donkey anti-rabbit
conjugated with Alexa Fluor 488;
                Stain 2: Rat anti-mouse antibody, and donkey anti-rat
conjugated Alexa fluor 594;   

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