cytochrome oxidase reaction in monkey brain tissue
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.
Is it better to react first and then mount on slides?
Any and all help would be greatly appreciated. Thanks to all in advance.
State University of New York
Upstate Medical University
Neurosurgery Lab, 4117 IHP
750 East Adams St.
Syracuse, NY 13210
e-mail: firstname.lastname@example.org or email@example.com
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