Blots were incubated in blocking buffer (p38α MAPKp44/42 MAPK (Erk1/2), After four washes with buffer, the blots were incubated with corresponding peroxidase-conjugated secondary antibody in corresponding blocking buffer for 2 h at room temperature.
After 5 times 5 min washes in blocking buffer, the blots were incubated for 1 hr in 2nd Ab linked to alkaline phosphatase (AP) diluted 1∶10,000 in blocking buffer.
Antibodies were diluted in the respective blocking buffer and blots were incubated O/N at 4°C.
After washes in blocking buffer, the blots were incubated simultaneously in 2nd Abs linked to Cy3 and Cy5.
Antibody dilution was prepared in blocking buffer and blots were incubated with monoclonal antibody overnight at 4°C.
After rinsing with the washing buffer, the blots were incubated with the secondary antibodies (either horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG; 1 2000; Dingguo Biotechnology) at room temperature for 45 min.
After washing (3 × 10 min) in TPBS buffer, the blots were incubated overnight with primary monoclonal antibodies against NMDAR (1 : 500) or mGluRs (1 : 1000) and subsequently, after washing with TPBS (3 × 10 min), with secondary antibodies conjugated with HRP (1 : 6000).
After washing (three times in TBST buffer), the blots were incubated for 1 hour with the secondary antibody [goat anti-rabbit IgG (H+L) HRP conjugate, Bio-Rad, #170-6515].
