Anti col6a3

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).

Immunoblotting was performed after 10% or 12% SDS-PAGE by probing with primary antibodies at the indicated dilutions: anti-GAPDH (1: 1000, mouse monoclonal; Milipore, Billerica, MA, USA), anti-COL6A3 (1:200, mouse monoclonal; Santa Cruz Biotech, Santa Cruz, CA, USA), anti-GSTP1 (1:1000, rabbit polyclonal; Abcam, Cambridge, UK), anti-FLNA (1:1000; rabbit polyclonal; Cell Signaling Technology, Danvers, MA, USA), anti-NCAM (1:10,000, rabbit polyclonal; Santa Cruz Biotech), anti-PLP1 (1:1000, rabbit polyclonal; Abcam), anti-SYNPO (1:500, rabbit polyclonal; Abcam), anti-VIM (1:1000, rabbit polyclonal; Genscript, Piscataway, NJ, USA). Twenty to fifty micrograms of proteins were used depending on the sensitivity of the specific antibody. Immunoreactivity was detected by using an HRP chemiluminescent substrate reagent kit (Invitrogen, Carlsbad, CA). A pooled sample was used to normalize the inter-gel variation between repeated runs for the same protein. Immunoreactivities of antibodies were visualized with Luminata™ Forte or Crescendo Western HRP substrate (Merck Millipore, Germany) and quantified with the Alliance 4.7 image analyser (UVItec, UK).

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).