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
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
Any suggestions would be greatly appreciated!
Stanford Neuroscience Program
Beckman B103, Stanford, CA 94305
phone (650) 736-1066/fax (650) 725-3958
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