Anti gad67

Mice were anesthetized with 2% pentobarbital and perfused with 0.01 m PBS, followed by fixation with 4% PFA. Coronal brain sections (30 µm) of the amygdala were incubated with PBS containing 0.1% Triton X‐100 for 20 min and blocked with PBS containing 5% goat serum (Gibco, USA) for 60 min. Then, the samples were incubated with the following antibodies and reagents: anti‐Dcn (1:500, Abcam, USA), anti‐CaMKII (1:500, Millipore, USA), anti‐Iba1(1:500, Abcam, USA), anti‐GFAP(1:500, Abcam, USA), biotinylated Wisteria floribunda lectin (1:1000, Sigma, USA), anti‐Gad67 (1:500, GeneTex, USA), anti‐Col6a3 (1:100, Santa Cruz, USA), streptavidin‐Alexa Fluor 594 (1:500, Abcam, USA), and antimouse/rabbit Alexa Fluor 594/488 (1:500, Invitrogen, USA). Images were acquired using an A1 Si confocal microscope (Nikon, Japan).

Western blotting was performed in accordance with standard procedures (Molecular Clone, Edition II). BLA tissues were collected as described above and homogenized in ice‐cold lysis buffer containing phosphatase and protease inhibitors. Twenty micrograms of total protein from the tissues was resolved by SDS‐PAGE and transferred to PVDF membranes (Millipore, USA). After blocking with 5% nonfat milk, the following primary antibodies were applied: anti‐NR1 (1:3000, Millipore, USA), anti‐Gad67 (1:1000, GeneTex, USA), anti‐NR2A(1:500, Proteintech, China), anti‐NR2B(1:500, Proteintech, China), anti‐NR3A(1:500, Invitrogen, USA), anti‐NR3B(1:500, Invitrogen, USA), anti‐Dcn (1:1000, Abcam, USA), anti‐Col6a3 (1:500, Santa Cruz, USA), anti‐Col17a1 (1:1000, Invitrogen, USA), anti‐β‐actin (1:5000, Cell Signaling, USA), and antimouse/rabbit secondary antibodies (1:5000, ABclonal, China). Images were captured using a Bio‐Rad imaging system (Bio‐Rad, China), and the bands were analyzed by ImageJ (Version 1.53C).