immunohistochemistry in thick brain sections combined with neuron tracing

Posted by David Stuss on
URL: http://confocal-microscopy-list.275.s1.nabble.com/immunohistochemistry-in-thick-brain-sections-combined-with-neuron-tracing-tp589948.html

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: [phone]
e-mail: [hidden email]