Mglur5

Total protein of the hippocampal tissues were extracted by the Total Extraction Sample Kit (Sigma, Shanghai, China) and the protein concentration was determined by the BCA assay (Bio-Rad Hercules, CA, USA). Two hundred micrograms of each sample protein were separated through SDS–PAGE electrophoresis utilizing a 10% running gel and then transferred to the 0.45 um polyvinylidene difluoride (PVDF) membrane (Millipore, Waltham, MA, USA). The membranes were then incubated for 2 h at room temperature in 5% fat-free dry milk in phosphate buffered saline with a 0.1% Tween-20 (PBST). Immunoblotting was carried out overnight at a temperature of 4 °C with the specific antibody titer in the serum of rabbits immunized with the purified protein mGluR1 was 1:3000 (Abcam, Cambridge, UK) and mGluR5 was 1:1000 (Abcam, Cambridge, UK) in PBST buffer. The membranes were stripped three times with PBS-Tween-20 0.1% and incubated with the appropriate HRP-conjugated goat antirabbit secondary antibody (1:10,000, Pioneer, Xi'an, Shaanxi, China) at room temperature for 2 h. After three rinsing cycles in the PBST buffer, protein bands were visualized with an enhanced chemiluminescence kit (Pierce, Holmdel, NJ, USA), and the lanes were calculated by the Image Lab software (Bio-Rad, Hercules, CA, USA).