Re: immunohistochemistry in thick brain sections combined with neuron tracing

Posted by Martin Wessendorf-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/immunohistochemistry-in-thick-brain-sections-combined-with-neuron-tracing-tp589948p589949.html

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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