Mouse anti galc

OPCs were isolated from mixed male and female P5-8 Adgrg1^+/+ or Adgrg1^-/- mouse forebrains as previously described (Watkins et al., 2008 (link); Wang et al., 2001 (link)). Briefly, OPCs were purified by negatively selecting with mouse anti-Thy1.2 (BioRad, Hercules, CA; Cat #MCA02R) and mouse anti-GalC (Millipore, Burlington, MA; Cat #MAB342), followed by mouse anti-O4 (O4 hybridoma supernatant) for positive selection. After releasing OPCs from the O4 plate by trypsinization, cells were resuspended in proliferation media containing PDGF-AA and NT-3 (PreproTech, Rocky Hill, NJ ). OPCs were plated on coverslips coated with poly-D-lysine before coating with laminin I (R and D Systems, Minneapolis, MN; Cat#3400-010-01) or fibronectin (Millipore, Burlington, MA; Cat#341668) as previously described (Dugas et al., 2006 (link)). After 24 hr, rTG2 (2 nM) was added to the cultures and incubated for an additional 24 hr before fixation with 4% PFA and staining for PDGFRα and Ki67.

The cells or frozen tissue sections were fixed with 4% paraformaldehyde (PFA) in PBS (Wako) for 15 minutes at room temperature, and permeabilized with 0.1% Triton X-100 (SIGMA-ALDRICH) for detection of intracellular proteins and a membrane marker NG2. To detect another membrane marker GalC, the permeabilization step was omitted. After blocking with Power Block Universal Blocking Reagent (BioGenex Laboratories) for 1 hour at room temperature, cells were incubated with primary antibodies overnight at 4 °C. The primary antibodies were as follows: rabbit anti-NG2 (1:250; Millipore), mouse anti-GalC (1:250; Millipore), mouse anti-A2B5 (1:300; abcam), rabbit anti-GFAP (1:1000; Thermo Scientific), mouse anti-S100 (1:1000; SIGMA-ALDRICH), mouse anti-APC (1:20; Millipore), mouse anti-NeuN (1:100; Millipore), and rabbit anti-Iba1 (1:250; Wako). After washing, the cells were labeled with secondary antibodies for 45 minutes at room temperature. The secondary antibodies were as follows: rabbit/mouse IgG-Alexa 594 (Life Technologies), mouse IgG- and mouse IgM-Alexa 647 (Life Technologies). The samples were mounted with Vectashield containing DAPI (Vector Laboratories).