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Cytochrome oxidase histochemistry

Vanderbilt vanderbilt.edu
Sun Jan 28 23:38:31 EST 1996

In article <udbl080.2.014123C8 at bay.cc.kcl.ac.uk>, udbl080 at bay.cc.kcl.ac.uk
(Phillip R. Gordon-Weeks) wrote:

> Can cytochrome oxidase histochemistry for cortical barrel fields be combined 
> with immunohistochemistry and if so can glutaraldehyde be used?

You certainly should be able to. Those of us who study the visual cortex
use CO histochemistry to show CO blobs in V1, and many papers have been
published showing the relationship of immunochemical labeling of
transmitters, calcium binding proteins, whatever, to the CO blobs. Just to
name a few, look for papers by Margaret Wong-Riley, Stuart Hendry, Anita
Hendrickson, Vivien Casagrande (my supervisor here at Vanderbilt), to see
how much glutaraldehyde you can use and still get good CO staining. Of
course, the "harder" the fix, the longer the longer the incubation time
you will need. You might want to use a nickel or cobalt enhanced recipe
(see below) instead of DAB alone as the chromagen, as these are supposed
to be more sensitive. 

Crockett, D.P., Maslany, S., Harris, S.L. Egger, M.D. (1993) Enhanced
cytochrome-oxidase staining of the cuneate nucleus in the rat reveals a
modifiable somatotopic map. Brain Research: 612:41-55

55 mg Diamino benzidine (DAB)
7.5 mg cytochrome c
5 g sucrose
0.25 ml dimethyl sulfoxide (DMSO)100 ml 0.1 M sodium phophate buffer (pH 7.6)
2.5 ml 1% cobalt chloride, added dropwise while stirring
2 ml nickel ammonium sulfate, added dropwise while stirring

Free floating sections are incubated in a 37 degree oven.

Using this recipe, these authors were able to get good CO staining in rats
perfused with 500 ml of 1% paraformaldehyde, 2% glutaraldehyde, which is a
strong enough fix for most any immunocytochemical purpose.

Jamie Boyd
Dept. of Cell Biology
Vanderbilt University
boydjd at ctrvax.vanderbilt.edu

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