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Cytochrome oxidase histochemistry

CROCKETTDP crockettdp at aol.com
Wed Feb 7 22:58:26 EST 1996


I am glad to see that one of our papers on CO histochemsitry made
newgroup.  We have since modified the protocol to be used with
immunohistochemistry.  We have found that using a modification of Berod's
pH-change method works quite well well: good CO activity and good
immunocytochemistry  on alternate sections.

The fixation is as follows (transcardial perfusion):
In adults rats:
1. 100 ml PBS wash  
2.  100-150 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (infant
rats 0.05 M buffer), pH 6.5
3.   250 ml of 4% paraformaldehyde in 0.1 M phosphate buffer plus 15%
saturated picric acid, pH 8.
3   250 ml 4% paraformaldehyde in 0.1 M phosphate buffer, pH 10.5, plus
15% sucrose. 

You could react the same day (best result) or postfix no more than a day
or so.

Reaction Medium (made fresh) with free-floating frozen sections (made ind
100 ml 0.1 M sodium phophate buffer (pH 7.6):

50 mg Diamino benzidine (DAB)
50 mg cytochrome c
5 g sucrose
0.25 ml dimethyl sulfoxide (DMSO)
2.5 ml 1% cobalt chloride, added dropwise while stirring rapidly
2 ml nickel ammonium sulfate, added dropwise while stirring rapidly.
incubate sections in 37 degree C oven 1 -2 hrs, with monitoring.


N.B.  We have found that cytochrome C may be increased to to as much as
200 mg/ 100 ml of reaction medium.  This seems to work quite well with the
above fixation protocol.

N.B.  We have also decreased the amount of heavy metal salts (to 0.7 ml of
each per 100 ml of reaction medium.

If you have any questions please contact me at the lab.  I would be glad
to send additional reprints.

Yours

David P. Crockett, Ph.D.
Department of Neuroscience and Cell Biology
University of Medicine and Dentistry
Robert Wood Johnson Medical School

Voice: 908-235-4522
FAX:   908-235-4029
E-mail (lab):  crockett at umdnj.edu

David P. Crockett, Ph.D.
Department of Neuroscience and Cell Biology
UMDNJ-Robert Wood Johnson Medical School
675 Hoes Lane
Piscataway, NJ 08854



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