Mab1326

The cultured cells were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) and used for immunostaining. Fixed cells were blocked with 5% normal goat serum in phosphate-buffered saline and 0.1% Triton X-100 (PBST) and then incubated with primary antibodies overnight at 4°C. The primary antibodies used were as follows: rat anti-GFP antibody (1/500, Nacalai Tesque, 04404-84), monoclonal anti-α-tubulin antibody (1/500, Sigma, T9026), rat anti-myelin basic protein (MBP) antibody (Millipore, MAB386), monoclonal O4 antibody (1/300, R&D systems, MAB1326) and rabbit anti-paxillin antibody (1/250, abcam, ab32084, clone Y113). After being rinsed with PBST, the cells were incubated with secondary antibodies. The secondary antibodies used were Alexa 488- or 594-conjugated goat anti-mouse, anti-rabbit and anti-rat IgG or goat anti-mouse IgM (Molecular Probes). Fluorescent signals were visualized using AX70 fluorescence microscope (Olympus, Tokyo) and A1Rsi confocal fluorescence microscope (Nikon, Tokyo). The number of OL primary processes was analyzed using “Analyze/Sholl” tool of Fiji software based on ImageJ (NIH).

Immunocytochemistry was performed essentially as described and involved cell fixation in 4% paraformaldehyde, permeabilization (except for O4 staining), blocking and consecutive incubation with primary and secondary antibodies, separated by extensive washing cycles (33 (link)). In addition to antibodies already mentioned for their use in immunohistochemistry, the following primary antibodies were used: mouse anti-O4 monoclonal (R&D systems, #MAB1326, Lot# HWW1115081), rabbit anti-Tcf4 (Abcam, #ab185736, Lot#GR3229215-1), rabbit anti-GFP antiserum (Molecular Probes, #A11122, Lot# 1293114), rat anti-MBP monoclonal (Abcam, #ab7349, Lot# GR188102-12 and Bio-Rad, #MCA409S, Lot #210610). Secondary antibodies were coupled to Cy3 (Dianova) or Alexa-Fluor (Molecular Probes) fluorescent dyes. Stainings were documented on a Leica DMI 6000B inverted microscope or a Zeiss Apotome.