Dear All,

I am trying to observe the phosphorylation state of a nuclear protein (CREB) 
in the brain, in mice. Briefly, I perfuse the animal with formaldehyde, 
cryoprotect in  sucrose, and then I cut 30 um slices and stain with a=20
phospho-specific antibody.

The problem is, the staining is very dim in cells that are located a few 
hundred microns from the surface of the brain (e.g., cells far away from the 
edges of each 30 um slice). I've tried 5 non-phospho-specific antibodies, 
which have no such problem. This suggests that this is a problem specfic to 
the phosphorylation state of the protein.

Does anyone know if protein phosphorylation state is labile during=20
perfusion? I am already doing very fast perfusions with a pump (15 ml/min 
for a mouse), preserving the brain in ice as soon as the blood runs clear, 
and postfixing. Should I perfuse with phosphatase
inhibitors?

Any suggestions would be greatly appreciated!

Best,
Ed

Ed Boyden
ed_boyden@hotmail.com
Stanford Neuroscience Program
Beckman B103, Stanford, CA 94305
phone (650) 736-1066/fax (650) 725-3958

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