Biocytin and double-labelling in EM

Markus Wallstein Markus.Wallstein at zfn.uni-bremen.de
Tue Nov 12 21:26:43 EST 1996


I am tracing tectum-neurons in the tectum of salamanders with the 
retrograde neuronal tracer biocytin. I did develop the biotin label in an 
preembedding procedure at 50 micrometer vibratome-slices with DAB as 
chromogen and heavy metal intensification. After I flatembedded the 
sections in epon I did the staining for e.g. GABA with an immunogold 
procedure. I did get considerable problems to find double-labellings for 
the tracer and GABA at all. I am afraid this is because of the very dense 
DAB precipitate in all labelled structures so that there is no 
possibility for the GABA antibody to bind at all. I am now planning to to 
develop the slices for both (biocytin and transmitter) in a postembedding 
immunogold-procedure with goldparticles in different sizes. I have 
allready found goldcoupled streptavidin but not avidin. I know that 
streptavidin is in order of several magnitudes less sensitive to biotin 
than avidin and I think this will work only as step in a multi-step 
method (marker for biotinylated primary antibodies). There is of cause 
also the possibility for buying anti-biotin antibodies but comparing 
with the ABC-method there is of cause a lack of sensitivity too. Does 
anybody have any experience with a postembedding immunogold-procedure for 
the detection of biocytin.

Thank you in advance for any hint.

Markus Wallstein



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