Odyssey system and software

Muscle tissues were homogenized in 1.4 mm Zirconium Beads Pre-Filled Tubes (OPS Diagnostics) with a MagNA Lyser (Roche Diagnostics). Samples were homogenized for 20 s at speed 7,000 (2–5 rounds) in 1 mL 125 mM Tris-HCl (pH 6.8) buffer supplemented with 20% (w/v) SDS. Protein concentrations were determined by the Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific, the Netherlands) using bovine serum albumin as a standard according to the manufacturer's instruction. Western blotting analysis was done as described previously [45 (link)], using GTX15277 as a primary antibody for dystrophin (diluted 1:2,000; Gene Tex) and AB72592 as a primary antibody for alpha-actinin (loading control, diluted 1:1,000; Abcam, United Kingdom). Secondary antibodies, IRDye 800 CW (1:5,000, immunoglobulin G; Li-Cor, NE) and IRDye 680TL (1:10,000 Li-Cor), were used for dystrophin and alpha-actinin, respectively. Membranes were analyzed with the Odyssey system and software (Li-Cor).

In a separate cohort of mice after one month of hypoperfusion (n = 8) or a sham (n = 5) procedure, after decapitation, the brain was rapidly removed, the cerebellum discarded and the remaining brain frozen in liquid nitrogen. Myelin-enriched fractions were prepared by sucrose density centrifugation [29] (link) and the total protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, UK). Proteins were separated by Bis-Tris 4–12% SDS-PAGE (NuPage® Novex®, Life Technologies) and transferred onto PVDF membrane (Immobilon-FL, Millipore). Immunobloting was performed using the Odyssey Infrared Imaging System (LiCor Biosciences, Lincoln, NE, USA). Membranes were blocked 1 hour at room temperature in Odyssey blocking buffer (diluted 1∶1 with phosphate-buffered saline), washed in phosphate-buffered saline–Tween (phosphate-buffered saline with 0.1% Tween) and incubated over-night at 4°C with MBP (1∶10.000, Millipore). After gentle washing, membranes were then incubated 1 hour at room temperature with GAPDH (1∶14.000, Sigma) which was used as a loading control. Membranes were then incubated for 45 minutes with the appropriate fluorescent secondaries (1∶3000, LiCor Biosciences). The western blots were analysed using the LiCOR Bioscience Odyssey system and software. The four MBP isoforms were analysed together and normalized to GAPDH.