Sections were then incubated for 1 hour over unconjugated goat anti rabbit IgG monovalent Fab fragments. Membranes were incubated over night in TBS T buffer containing the right primary antibodies and natural compound library 50-degree BSA. Matching anti rabbit HRP or anti goat HRP and ECL Advance Western Blotting package were used for detection. Blots were washed 4 times for five minutes each with TBS T between blocking and applications of antibodies. Blots were scanned and densitometry was performed via Image J. Serine/threonine phosphatase task assay products were purchased from Promega Corp.. Assays were performed on a 96 well plate format, per manufacturers guidelines. Shortly, to remove endogenous phosphates and phosphatase inhibitors from hippocampal RIPA lysates, samples were desalted utilising the Zeba micro spin desalting columns. Each sample was run in responses, each contained 5 ul of just one mM phosphopeptide, 10 ul of proper 5 phosphatase response load, 2 ul of lysates, and 33 ul of deionized H2O. Protein phosphatase 2A effect buffer contained 250 mM imidazole, 1 mM EGTA, 0. Hands down the W mercaptoethanol, and 0. 5 mg/ml acetylated BSA. As well as the reagents listed for PP2A reaction buffer, PP2B reaction buffer also involved 50 mM MgCl2, 5 mM NiCl2, 250 ug/ml calmodulin. Plates were incubated at 30 C for 30 minutes for phosphatase reactions to happen. Reactions were stopped by addition of 50 ul of Molybdate Dye/Additive mixture to each well. Dishes were subsequently incubated at room temperature for thirty minutes to permit the Molybdate Dye to bind to free phosphates released from the reaction. Plates were read using a plate reader with 630 nm filter. Optical densities of the samples were determined buy Cathepsin Inhibitor 1 based on the optical densities of free phosphate standards. Specific activities for PP2A and PP2B were expressed as pmol phosphates per minute per ug of total protein. Immunohistochemistry was performed as previously reported. Rats were killed at 24 hours post TBI, their heads were set for 24 hours in 401(k) paraformaldehyde and cryoprotected in 30 % sucrose for 2 days before sectioning to 50 um thick slices with a sliding microtome. One more blocking step for 1-hour with unconjugated anti mouse IgG monovalent Fab fragments was performed following blocking with serum, to cut back back ground staining on injured tissues when staining with monoclonal PHF1 antibody. For double labelling of phospho tau and activated JNK, sequential applications of primary antibodies were employed. First, sections were incubated with rabbit anti pS199, followed closely by goat anti rabbit secondary antibody conjugated to Alexa Fluor 488. Parts were blocked again for thirty minutes with three minutes normal rabbit serum to saturate available binding web sites around the first secondary antibody with IgG. So that the second extra antibody wouldn’t bind to it this was done to protect the rabbit IgG. Rabbit anti g JNK was subsequently used, followed closely by goat anti rabbit conjugated to Alexa Fluor 594.