Western Blot Analysis of Microglia and Macrophage Proteins

Acutely isolated and cultured microglia or human macrophages were lysed in STET lysis buffer supplemented with protease and phosphatase inhibitors (Sigma Aldrich). Lysate protein content was quantified using Bradford assay (Biorad) according to the manufacturer´s protocol. At least 10 μg per sample were separated on a bis-tris acrylamide gel followed by western blotting either on a PVDF or nitrocellulose membrane (Millipore) using the following antibodies: Iba1 (1:1000, Wako); GFAP (1:1000, Dako); Tuj1 (1:1000, Covance); CNPase (1:1000, Abcam); NPC1 (1:1000, Abcam); NPC2 (1:1000, Sigma Aldrich); LAMP1 (1:1000, Sigma Aldrich); Cathepsin B (1:2000, R&D System); Cathepsin D (1:1000, Novus Biologicals); mouse CD68 (1:1000, AbD Serotec); human CD68 (1:500, Acris); mouse CD63 (1:1000, Abcam); mouse GRN (1:50, clone 8H10,^{85 (link)}); human GRN (1:1000, Invitrogen); TREM2 (1:10, clone 5F4^{66 (link)}); APOE (1:1000, Millipore); PLP1 (1:1000, Abcam); EGFR (1:1000, Abcam) and TGFB1 (1:1000, R&D System). Blots were developed using horseradish peroxidase-conjugated secondary antibodies (Promega) and the ECL chemiluminescence system (GE Healthcare). An antibody against Calnexin (1:1000, Stressgen) or GAPDH (1:2000, Abcam) was used as loading control. Blot densitometry quantification was performed using gel analyzer tool in ImageJ (NIH) with at least n = 3 for both human and murine samples.