Dear Dori,

We regularly do CO reactions on tangential sections of wallaby brain (cortex).
These are pouch young so the size of brain is about 1-2 cm3. You may 
need to dissect out the piece of tissue you are interested in first.

We perfuse the animal briefly with 0.9% saline then 4% 
paraformaldehyde in 0.1M PB. The brain is dissected out and post 
fixed in 4% paraformaldehyde for 1 hour at 4 degrees celsius followed 
by cryoprotection in 30% sucrose/0.1M PB overnight at 4 degrees or 
until the tissue sinks. We use 30um frozen sections adhered directly 
onto gelatinised slides (2%) which are then air dried overnight. The 
CO reaction usually takes about 3 days. I check them daily and stop 
the reaction when the product is dark enough.

Also mark the piece of tissue before cutting, I make distinctive cuts 
on two surfaces (ie anterior and ventral) so the sections can be 
orientated later. The reaction can be speeded up by heating but it's 
harder to judge the endpoint.

Hope this helps,

Cheers, Cath


At 3:27 PM -0400 10/6/03, DoriPJ@aol.com wrote:
>Hello Histonetters,
>
>I am searching for the best protocol for doing cytochrome oxidase 
>(CO) reaction in monkey brain tissue.  I have tried it free-floating 
>and mounted on subbed slides, and have had problems with both 
>methods.  The mounted sections tended to fall off the slides in the 
>reaction bath, also had problems with the entire glass staining, and 
>this method takes forever (even with heating and shaking).  When we 
>tried the free-floating method, it seemed to work a little better, 
>but still took a very long time, and then we had the problem of 
>piecing together the sections.  We are doing flattened, tangential 
>sections of visual cortex on a sliding microtome (frozen sections, 
>30um thick).  Due to the flattening process, we usually end up with 
>3 pieces per section that are often difficult to orient correctly.
>
>Amount of fixation also seems to be an issue.  Some tissue that we 
>tried that wasn#226#Aot well fixed produced crummy looking sections 
>(shreddy edges) but showed CO activity.  Other tissue that was well 
>fixed sectioned fine but CO showed nothing.
>
>So, what I need is advice from a technician:  all the tips and 
>tricks that techs know but are never in the published protocols. 
>Specifically:
>     How much fixation?
>How to speed up the reaction time? (Our tissue literally took 
>8-12hrs to develop at all!  Some sections we left in almost 24hrs!)
>How to keep sections from lifting off slides in reaction bath.
>Or
>Is it better to react first and then mount on slides?
>
>Any and all help would be greatly appreciated.  Thanks to all in advance.
>
>
>Dori Joiner
>State University of New York
>Upstate Medical University
>Neurosurgery Lab, 4117 IHP
>750 East Adams St.
>Syracuse, NY 13210
>Phone: 315-464-5498
>FAX:    315-464-5504
>e-mail: joinerd@mail.upstate.edu   or   doripj@aol.com
>
>                            
>
>

-- 
---------------------------------------------------
Catherine Gorrie
School of Medical Sciences,
University of New South Wales
Sydney, N.S.W. 2052

Phone: 61-2-9385 2462
Fax  : 61-2-9385 8016
e-mail: c.gorrie@unsw.edu.au