Mouse anti mog

To identify myelin lineage cell subsets, total brain cells were labeled with anti-human PE-conjugated mouse anti-O4 (Miltenyi Biotec, Auburn, CA), APC-conjugated mouse anti-A2B5 (Miltenyi), mouse anti-MOG (Millipore, Temecula, CA), V450-conjugated rat IgG1 (BD Biosciences, Mississauga, ON), or concentration-matched isotype controls at 4°C in 1 mmol/L ethylenediaminetetraacetic acid (EDTA)/fetal bovine serum supplemented phosphate-buffered saline (FBS-PBS). Immunolabeled neural cells were analyzed for expression of the surface markers on BD FACSAria™ II (BD Biosciences) (equipped with 405 nm, 488 nm and 633 nm lasers) using BD FACSDiva™ 6.1.3 software (BD Biosciences). To gate for viable cells, propidium iodide (Invitrogen) was added to the labeled cell samples just before sorting.

The protein concentration of each sample was measured using the Lowry method and the colorimetric reaction was measured on a SpectraMax M3 plate reader (Molecular Devices). 10–40µg of total protein was loaded on mini Protean TGX Precast gels (Biorad). After electrophoresis, proteins were blotted on a PVDF membrane (Biorad) and blocked in 5% Blotting Grade Blotter (Biorad) diluted in Tris buffered saline (TBS) containing 0.1% Tween 20 (TBST). Blots were then incubated with either of the following antibodies: chicken anti-human MCT1 antibody, mouse anti-PLP (MilliporeSigma), mouse anti CNPase (MilliporeSigma), rabbit anti-MCT2 (MilliporeSigma), rabbit anti-GLUT1 (abcam), rabbit anti-GLUT3 (abcam), mouse anti-MBP (Biolegend), mouse anti-MOG (MilliporeSigma), rabbit anti-Connexin-43 (MilliporeSigma), mouse anti-GAPDH (Thermo Scientific), and mouse anti-MAG (MilliporeSigma)(for more details see Key Resources Table). Membranes were overnight incubated with primary antibodies at 4°C. Membranes were washed with TBST and incubated with HRP labeled secondary antibodies for 1 hour at room temperature. After two washes in TBST and one wash in TBS, membranes were exposed to ECL reagent for 2 min. (Thermo Scientific) and chemiluminescent signal was detected with the LAS Imager 4000 (GE Healthcare).