Mouse anti olig1

Immunofluorescence was performed as described previously [22 (link)]. After blocking with 10% normal goat serum (Sigma-Aldrich, USA), slices were incubated with primary antibodies as following: rabbit anti-GFAP (1:300; Cell Signaling Technology, USA), rabbit anti-IBA1 (1:400; Abcam, Cambridge, MA, USA), rabbit anti-beta Amyloid (1:50; Abcam, Cambridge, MA, USA), and mouse anti-Olig1 (1:100; Santa Cruz, USA). After incubated overnight at 4 °C and washed three times in 0.01 M PBS (3 × 5 min), slices were incubated with the following secondary antibodies for 1 h at room temperature: Alexa Fluor^® 594 conjugated goat anti-rabbit IgG or Alexa Fluor^® 488 conjugated goat anti-rabbit IgG or Alexa Fluor^® 488 conjugated goat anti-mouse IgG(1:1000; Cell Signaling Technology). Slices were mounted in ProLong^® Gold antifade reagent (Thermo Fisher Scientific, USA) prior to imaging.

Approximately 30 μg of total protein was loaded on an SDS–polyacrylamide gel as described previously [22 (link)]. The primary antibodies were used as follows: rabbit anti-myelin basic protein (MBP) and rabbit anti-IRAK1 (1:1000; Abcam, Cambridge, MA, USA), mouse anti-IL-6 (1:500; Abcam, Cambridge, MA, USA), mouse anti-Olig1 and mouse anti-TNF-a (1:300; Santa Cruz, USA), mouse monoclonal anti-β-actin (1:3000; Cell Signaling Technology, USA). Membranes were incubated with the secondary antibodies for 1 h at room temperature: horseradish peroxidase (HRP)-conjugated goat anti-rabbit or HRP-conjugated goat anti-mouse IgG antibody (1:3000; Cell Signaling Technology, USA). The bands were quantified by densitometry with ImageJ software (ImageJ 1.4, NIH, USA).