Goat anti mouse igg h l alexafluor 488

Neurobasal A (w/o phenol red), supplement B27 (w/o antioxidants), high glucose DMEM (Dulbecco’s Modified Eagle’s Medium), FluoroBrite™ DMEM, heat-inactivated FBS (fetal bovine serum), 0.25% trypsin/EDTA and penicillin/streptomycin solution were obtained from Gibco (Life Technologies Ltd., Paisley, UK). The Cytotoxicity Detection Kit and Cell Proliferation Reagent WST-1 were purchased from Roche Diagnostics GmbH (Mannheim, Germany). Caspase-3 (Ac-DEVD-AMC) fluorogenic substrate was from Enzo Life Sciences (New York, NY, USA). CM-H₂DCFDA, fluorescently labeled secondary antibodies (goat anti-mouse IgG (H+L) AlexaFluor^® 488 and donkey anti-rabbit IgG (H+L) AlexaFluor^® 568)) and ProLong^®Gold antifade reagent were from Molecular Probes (Life Technologies Corporation, Eugene, OR, USA). Cannabidiol (CBD, 2-[1R-3-methyl-6R-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol) was purchased from Cayman Chemical (Ann Arbor, Michigan, USA). All other reagents were from Sigma (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany).

SK-N-SH cells grown as a monolayer in coverslips were fixed in 4% formalin. Fixed cells were permeabilized in PBS/Triton X-100 0.1% and blocked in PBS/2% BSA. Cells were stained with primary antibodies: 1:400 anti-IGF2R (rabbit monoclonal, Abcam 124767) and 1:250 anti-GABBR1 antibody (mouse monoclonal, Abcam ab55051) overnight at 4°C. After three washes in the wash buffer (PBS/2% BSA), the cells were incubated with secondary antibodies (goat anti-mouse IgG [H+L] AlexaFluor 488 [Molecular Probes A-11001], donkey anti-rabbit IgG [H+L] AlexaFluor 647 [Abcam 150075]) diluted 1:500 for 1 h at room temperature. The coverslips were washed twice in wash buffer and three times in PBS and mounted on a microscope glass in SlowFade with DAPI (Molecular Probes) before images were captured with Leica SP5/DM6000 confocal microscope.