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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

David-

My wife and a grad student studied the dendritic arbors of L4 stellate 
cells in thick slices of cat cortex.  The cells had been injected in 
fixed tissue (biotinylated-LY + ABC or LY w/ fluorescence).  They had 
hoped to combine with IHC, but the arbor project became so time 
consuming that they never got to that.  Data papers were never published 
because the student finished an extensive thesis and then went on to 
other things, and my wife passed away.  They did, however, publish a 
methods paper that might provide some hints for you: Pace CJ, Tieman DG 
& Tieman SB, Intracellular injection in fixed slices: obtaining complete 
dendritic arbors of large cells, J Neurosci Methods, 119 (2002) 23-30.

-dave

David Stuss wrote:
> Search the CONFOCAL archive at 
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I was wondering 
> if anyone has developed an immunohistochemistry protocol for thick brain 
> sections.  I'm interested in tracing full dendritic arbors of single 
> dye-injected cortical neurons (in PFA-fixed mouse brain) and hence 
> require sections 200-300 um thick. My aim, if at all possible, would be 
> to differentiate cell subtypes by immunolabeling deep in the tissue 
> slice, either to identify previously dye-filled neurons, or to identify 
> neurons for tracing. 
> 
> This is a bit of a tall order but I would appreciate any input, in 
> particular regarding immuno conditions (detergents, temperatures, 
> incubation times for primary and secondary antibodies) and the depth of 
> penetration achieved. Input on combining IHC with neuron tracing in 
> fixed tissue would also be greatly appreciated. 
> 
> Thanks for your consideration,
> 
> David Stuss
> PhD Candidate, Michael Smith Foundation for Health Research
> University of Victoria, Department of Biology
> phone: (250) 472-5656
> e-mail: [log in to unmask] <mailto:[log in to unmask]>

-- 
David G. Tieman, PhD
Technology coordinator
Department of Biological Sciences
University at Albany, State University of New York
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Ph: 518-442-4317