Western Blot Analysis of Cell Markers

Cultured cells were processed for Western Immunoblotting analysis using a previously established protocol (Santa-Cecilia et al., 2016 (link)). Briefly, cells were recovered with M-PER buffer (Invitrogen) containing protease and phosphatase inhibitor cocktail (Invitrogen), and aliquots were resuspended for protein quantification with a Nanodrop 8,000 Spectrophotometer (Thermo Fisher Scientific). Then, protein extracts were resolved on a 4–12% SDS-PAGE gel and transferred to PVDF membranes. Membranes were then incubated with primary antibodies against GFAP (1:1,000, overnight; Wako), Iba-1 (1:500, overnight; Dako) and GAPDH (1:2000, 2 h; Sigma-Aldrich) and washed with Tris-buffered saline containing 0.1% Tween-20 before incubation with the rabbit secondary IRDye antibody (LI-COR Biosciences, Lincoln, NE, USA). Blots were imaged through the LI-COR Odyssey infrared imaging system (LI-COR Biosciences), and quantitative analysis was performed with the ImageJ software (dos Santos Pereira et al., 2015 (link)).