immunohistochemistry in thick brain sections combined with neuron tracing

[David Stuss]

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]

[Martin Wessendorf-2]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

David Stuss wrote:

> 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.

Dear David--

What you want to do may be trivial or it may be impossible: in my
experience it depends largely on the particular primary antibody.  Some
antibodies penetrate easily through tissue; others penetrate almost not
at all.  Try yours and see what happens.

Running your tissue through a freeze-thaw cycle will help penetration,
as will use of detergent in at least some cases.

In my experience, fluorophore-labeled streptavidin penetrates tissue
very easily, thus labeling a neuron filled with (say) Neurobiotin or
biocytin should be easy.

Good luck!

Martin Wessendorf

--
Martin Wessendorf, PhD   [phone] (office)
Associate Professor      [phone] (lab)
Dept Neuroscience      [phone] (FAX)
Univ Minnesota        e-mail: martinw(at)umn.edu