Mouse anti cb

Cells were seeded on 12-mm diameter glass coverslips and when they had reached approximately 60–70% confluence, cells were fixed for 30 min with 4% paraformaldehyde, blocked with TBS-containing donkey serum (10%) and incubated overnight at 4 °C with mouse anti-CB (1:1000, Swant) and rabbit anti-FAK (1:50; Cell Signaling Technology, Danvers, MA, USA) antibodies diluted in Tris-buffered saline (TBS 1X). After washing, cells were incubated for 3 h at room temperature with the following secondary antibodies: Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:100; Jackson Immunoresearch Laboratories, West Grove, PA, USA) and Cy3-conjugated donkey anti-mouse (IgG) (1:100; Jackson Immunoresearch Laboratories). 4′,6-diamidino-2-phenylindole (DAPI; 5 μg/mL; Molecular Probes, Eugene, OR, USA) was used to stain nuclear DNA and coverslips were mounted with Hydromount solution (National Diagnostics, Atlanta, GA, USA). Images were acquired using a LEICA fluorescent microscope DM6000B (Wetzlar, Germany) equipped with a Hamamatsu camera C4742-95 (Bridgewater, NJ, USA).

P14 BTBR and WT mice were decapitated, and their cerebella were dissected in ice-cold PBS. The total protein was extracted immediately, and protein concentrations were measured using a Bicinchoninic Acid Kit (Beyotime, China) as previously described (Xiao et al., 2017 (link)). The total protein (20 μg) of each sample was separated by 10% SDS-polyacrylamide electrophoresis (80 V, 100 min) and then transferred to a polyvinylidene fluoride (PVDF) membrane (220 mA, 60 min). The membranes were washed in 1% Tween-20/Tris-buffered saline (TBS) (TBS-T), blocked in 5% BSA/TBS-T (RT, 2 h), and incubated with a primary antibodies (4°C, 12 h) (1) mouse anti-CB (1:2000, Swant, Switzerland) and (2) mouse anti-GAPDH (1:2000, Cell Cwbio, China), followed by peroxidase-conjugated goat anti-mouse secondary antibody IgG (1:1000, Santa Cruz Biotechnology, United States). Bands were visualized using the chemiluminescence detection kit (Pierce, United States) under a Gel-Pro analyzer (Bio-Rad Laboratories, United States). Band intensity was quantified in Image Lab (Bio-Rad Laboratories, United States), and calbindin protein was normalized to GAPDH.