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]

[Dave Tieman]

Search the CONFOCAL archive at
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: [phone]
> e-mail: [hidden email] <mailto:[hidden email]>

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

[mmodel]

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

David - There are some clearing protocols that make tissues more transparent. Never tried any of them myself but I know that such things exist. You can try searching them in the literature or online.  

 

Michael Model, Ph.D.

Confocal Microscopy Core

Dpt. Biological Sciences

Kent State University

Kent, OH 44242

tel. [phone]

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Stuss
Sent: Monday, April 21, 2008 11:14 PM
To: [hidden email]
Subject: immunohistochemistry in thick brain sections combined with neuron tracing

 

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]