Tissue and blood collection

For immunohistochemistry, mice (5-6 mice/genotype/age-group/treatment) were anesthetized and transcardially perfused with 0.9% NaCl, followed by 4 % paraformaldehyde in phosphate buffer (pH 7.2 at 4°C), the brains and peripheral tissues carefully removed and post-fixed for 2-4 hrs, in 4% paraformaldehyde in phosphate buffer (pH 7.2) and placed in 15% sucrose in the solution of phosphate buffer overnight at 4°C.

Blood was collected into EDTA-treated tubes and serum was separated by centrifugation and stored at -20C.

For gene expression and protein analysis, animals were sacrificed by cervical dislocation, the brains and peripheral tissues were quickly isolated and then stored at -80°C until assayed, the tails collected from each mouse were genotyped through PCR followed by electrophoretic analysis, as described in the manufacturer’s instructions (Jackson Laboratory).