Mapn50

Cells (1 × 10⁶) were washed twice with HEPES-buffered saline, treated with Accutase (ThermoFisher, A1110501), and fixed for 20 minutes at room temperature in 2.0% PFA. Cell membranes were permeabilized by adding 0.1% TritonX-100 in PBS for 20 minutes at room temperature. Cell pellets were collected and incubated with primary antibodies diluted in blocking solution: mouse anti-Olig2 (1:200; Merck Millipore, MAPN50), mouse anti-beta III tubulin (1:150; Merck Millipore, MAB1637), mouse anti-GFAP (1:200; Merck Millipore, MAB360), rabbit anti-TTR (1:300;; ABBIOTC, 250892) and incubated overnight at 4 °C. After washing, secondary antibodies anti-rabbit Alexa Fluor-488 (Invitrogen, A-11008) or anti-mouse Alexa Fluor-555 (Invitrogen, A-21427;), were diluted in PBS and incubated for 1.5 hours at room temperature. Flow cytometric evaluation was conducted within 5 minutes. Cells were analyzed by flow cytometry using an EPICS XL-MCL FACScan (Becton–Dickinson, Mountain View, CA, United States).

Purified OPCs were cultured and grown to 80% confluence in 8-well clear tissue culture Millicell EZ SLIDES (Merck Millipore, PEZGS0816). Cells were fixed in 2% PFA for 10 minutes at room temperature, then the cells were incubated for 2 hours in blocking buffer (1% BSA, 4% normal donkey serum and 0.1% triton X-100 in PBS). Primary antibodies were diluted in blocking solution: mouse anti-Olig2 (1:150; Merck Millipore, MAPN50), rabbit anti-NG2 (1:200; Merck Millipore, AB5320), mouse anti-PDGFRα (1:200; Merck Millipore, 05–1135), mouse anti-sox2 (1:100; MAB4423A4; Merck Millipore), mouse anti-nestin (1:200; Merck Millipore, MAB353), mouse anti-beta III tubulin (1:100; Merck Millipore, MAB1637), rabbit anti-MBP (1:300; Merck Millipore, AB980), mouse anti-GFAP (1:200; Merck Millipore, MAB360), rabbit anti-TTR (1:200; ABBIOTC, 250892;) and incubated overnight at 4 °C. After washing, secondary antibodies anti-rabbit Alexa Fluor-488 (Invitrogen, A-11008) or anti mouse Alexa Fluor-555 (Invitrogen, A-21427) were diluted in PBS and incubated for 1.5 hours at room temperature. Sections were then washed 3 times in PBS before being counterstained with 4′,6′-diamidinio-2- phenylindole (DAPI) and mounted with ProLong Gold Antifade mountant reagent (Invitrogen, P36930). Samples were examined using confocal microscopy under a x40 objective lens (E800; Nikon, Champigny-sur-Marne, France, PCM2000).