Ab109746

Total protein was extracted using a commercially available mirVana PARIS RNA and Native Protein Purification kit (Thermo Fisher Scientific). Protein levels were detected using appropriate primary antibody dilutions against MOG (1:1000; sourced from Abcam, ab109746) and secondary antibody (Alexa 594-conjugated antibody to rabbit, 1:10 000, Life Technologies, Thermo Fisher Scientific). Primary antibody against alpha tubulin (sourced from Sigma-Aldrich, Cat# T5168) was used to determine equal protein loading. Quantification of protein bands was analysed using Image Studio Lite (LI-COR, Lincoln, NE, USA). Significance was determined by a one-way analysis of variance.

To quantify protein amounts, 2-month-old mice were intracardially perfused with 1× PBS using a peristaltic pump (World Precision Instruments) for 3 min. Then, the spinal cord was removed and dissected to isolate the lumbosacral ventrolateral white matter, which was subsequently flash-frozen on dry ice. The tissue was lysed with fresh SDS lysis buffer with Mini Complete protease inhibitor and PhoStop phosphatase inhibitor (Roche) and homogenized with Precellys 24. A final protein concentration of 1 mg ml⁻¹ was prepared in Laemmli buffer and 10 µg of protein was loaded per slot. The proteins were separated on a 12% Bis-Tris mini protein gel and run for 10 min at 80 V and 45 min at 200 V. Subsequently, proteins were transferred onto a PVDF membrane and detected using antibodies (MOG: Abcam, ab109746, rabbit, 1:2,000; secondary HRP-anti-rabbit, Cell Signaling, 7074 S, 1:15,000; MBP: Sigma, Atlas, AMAB91064, mouse, 1:5,000; secondary HRP-anti-mouse, Cell Signaling, 7076, 1:5,000; ACTB: Sigma, A5441, mouse, 1:2,500; secondary HRP-anti-mouse, Cell Signaling, 7076, 1:5,000). The antibodies were incubated separately and subsequently stripped using glycin, SDS and Tween for 10 min before proceeding with the next antibody. Detection was performed using ECL.

IHC was performed on formalin-fixed paraffin-embedded 5 μm–thick sections with EnVisionTM FLEX IHC system (DAKO). Steam antigen retrieval with citric acid buffer (pH 6.0, DAKO) was performed for MAG, MOG, and C9neo staining. Primary antibodies were incubated at 4°C overnight to identify MAG (1:1,000, ab89780, Abcam), MOG (1:1,000, ab109746, Abcam), PLP (1:500, MCA839G, Serotec), C9neo (1:200, monoclonal B7 and polyclonal, from Paul Morgan, Cardiff, United Kingdom), and CD68 (1:100, M0814, DAKO).