Ab40390

Frozen sections were blocked in PBS + 0.1% Triton X-100 (PBT) for 1 h. Primary antibodies were diluted in blocking solution (PBT + 1% BSA + 0.15% glycine) as follows: cleaved-Caspase-3 (casp3, D175, lot 43, rabbit, Cell Signaling Technologies, 1:200 dilution), Mbp (SMI 94R-0100 lot E10172EF, mouse, Covance, 1:1000 dilution), GFP (A11122, lot 1891900, rabbit, Invitrogen, 1:1000 dilution), CD45 (14-045-185 C30F11, Rat, Invitrogen, 1:100 dilution) and F4/80, CD11b, CD68 cocktail (MCA497, MCA74G, MCA1957, Rat, Bio-Rad, 1:200 dilution) and incubation performed overnight at 4 °C. After several washes, slides were incubated with secondary antibody (anti-rabbit alexaFluor555, anti-mouse alexaFluor488, anti-rat Cy3 or anti-rabbit alexaFluor488, Invitrogen, 1:500 dilution each) for 2 h before final washing. For dorsal root ganglia (DRGs) cultures, cells were fixed in 4% PFA for 10 min and immunostained using the same procedure with TuJ1 (MMS-435P, lot TU17044, mouse, Eurogentec, 1:1000 dilution) and Mbp (ab40390, Rabbit, Abcam, 1:200 dilution). Secondary antibodies used were as follows: anti-mouse alexaFluor 488 and anti-rabbit alexaFluor 555 (Invitrogen, 1:500 dilution each). Preparations were then mounted using Vectashield containing DAPI (Vector laboratories) and observed using Spinning Disk from Zeiss.

To access protein expression level of MBP in BACHD rats, western blot was applied using striatal brain lysis of male BACHD TG5 rats and WT controls at 3 months of age (n = 3). The striata were stored at −80 °C after dissection and were homogenized with a tissue homogenizer at a speed of 30 000 rpm for 30 s in 10 volumes (w/v) modified RIPA buffer (50 mm Tris pH 7.5, 150 mm NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS) with Complete Protease Inhibitor Cocktail tablets (Roche, Germany). After a further 5-min sonication with bath sonicator for shearing genomic DNA, the lysates were centrifuged at 4 °C for 15 min at 16 200g, and the supernatant was used for western blot analysis (52 (link)). The blot was probed with polyclonal rat antibody recognizing rat MBP (1: 5000, ab40390, abcam, USA) followed by incubation in the HRP-conjugated secondary antibody (1:10 000, ab191866, abcam, Cambridge, MA, USA). Finally, blots were developed with ECL Western Blotting Detection Reagent (RPN2134, Amersham Biosciences, Germany) and followed by visualization on X-ray film (AGFA, Germany).

Newborn double-homozygous Shi (Mbp^Shi/Mbp^Shi) mice (The Jackson Laboratory, Bar Harbor, ME, United States) were injected with dissociated single OPCs. The protocol followed previous work with minor modifications (Zhang et al., 2019 (link)). Briefly, to generate the lentivirus expression of mCherry protein, the backbone of pCDH-CMV-MCS-EF1-copGFP (System Biosciences) was used. Subsequently, the copGFP was replaced by mCherry and puromycin was inserted into the multiple clone sites. The positive plasmid was confirmed by sequencing and using for lentivirus packaging. OPCs were transfected with lentiviral expressed mcherry, and positive cells were selected by puromycin. Then, mcherry OPCs⁺ were transplanted bilaterally in the corpus callosum of 1 week-old Shi mice (2.0 × 10⁵ cell/mice). PBS-treated age-sex-, strain-matched mice, and sham control mice were used. After 5 weeks from the treatment, mice were anesthetized, and brains were isolated and fixed with 4% paraformaldehyde and cryoprotected using 30% sucrose. CNS coronal sections were stained with myelin basic protein (MBP) antibody (ab40390, Abcam) for evaluating the differentiation process of mcherry OPCs⁺. Tremor (Trembling time/total time) was analyzed for evaluating disease development. Kaplan-Meier analysis was used to assess the survival rate as described (Shinkai et al., 1992 (link)).