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Neuropeptide double labelling/two rabbit primaries.

M. Ferber Ferber at zoology.uni-frankfurt.de
Wed Jul 5 04:12:32 EST 1995


rambis at iastate.edu (Paul L Pearson) wrote:
>
>I am currently looking into doing double-labelling of pig brain tissue
>for neuropeptides using two rabbit antibodies and a two fluorescence
>detection system.  I am considering using unlabelled fab fragments to
>hide the first primary.  
>
>
>If anyone has experience (either good or bad) with this technique, I
>would appreciate references and/or advice that could make this job a
>little easier.  Thanks.  
>
>Please send responses to: rambis at cvm01.vm.iastate.edu
>


Hi Paul,
I do not have a direct experience with this type of double labelling experiments, but a 
colleague (which has left the scientific area) developed a method during which uses a 
biotinylated and a unlabelled primary antiserum. Thze biotinylated antiserum was labelled with 
flourescence labelled avidin whereas the other antiserum was labelled with an ordinary 
flourescence labelled secondary antiserum. The Method was described in:

Würden S., Homberg U., (1993) A simple method for immunofluorescent double staining with 
primary antisera from the same species.. J. Histochem. Cytochem. 41:627-630

Tho Co Author is now at the University of Regenburg in Germany, but I do not have his email 
adress.

The labelling of the antiserum with biotin is described in:

Antibodies
A laboratory manual
E. (?) Harlow, D. Lane
Cold Spring Harbour Laboratory 1988


The results achived with this method were really impressive. 


Good luck
Michael




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