I have used western blot to detect phosphoproteins and am aware that it is important to treat samples with phosphatase inhibitors (Sodium fluoride, Orthovanadate etc.) to stabilize phosphorylation. Can someone comment on ways to preserve phosphorylation state in histological samples?
I'm working 20 uM spinal cord slices that were perfused with 4% PFA in 0.1M PBS and cryopreserved in sucrose solution. The tissue has been mounted on Superfrost slides and stored at -20. At the moment I'm interested in fluorescent staining of phosphorylated NMDA receptor subunits. The antibody's spec sheet indicates that citrate-buffer retrieval is helpful but I am curious if there are other manipulations that can help preserve this type of post-translational modification.