Western Blot Analysis of Spinal Cord

Mice were transcardially perfused with cold phosphate-buffered saline (PBS) and then the spinal cords were isolated from spinal columns for western blot analysis. Western blots were performed using WES, an automated capillary-based size sorting system (ProteinSimple, San Jose, CA, USA)^{14 (link)}. All procedures were performed according to the manufacturer’s protocol. Briefly, 8 μL of diluted protein lysate was mixed with 2 μL of 5× fluorescent master mix and heated at 95 °C for 5 min. The samples, blocking reagent, wash buffer, primary antibodies, secondary antibodies, and chemiluminescent substrate were dispensed into designated wells in a microplate provided by the manufacturer. The plate was loaded into the instrument and protein was drawn into individual capillaries on a 25 capillary cassette provided by the manufacturer. Protein separation and immunodetection was performed automatically on the individual capillaries using default settings. The data was analyzed with Compass software (ProteinSimple). Primary antibodies used were anti-Iba1 antibody (abcam, ab153696), anti-βIII Tubulin antibody (abcam, ab78078), anti-GFAP antibody (abcam, ab4674), and anti-Olig2 antibody (abcam, ab109186).