Pa5 95505

Frozen sections were rinsed in phosphate-buffered saline (PBS) and then blocked in PBS containing 0.3% Triton X-100 and 1% donkey serum for 30 min. The sections were incubated with the primary antibodies at 4°C overnight. The following primary antibodies were used: anti-NeuN (1:100, ab177487, Abcam), anti-GFAP (1:400, ab4674, Abcam), anti-C3 (1:300, ab200999, Abcam), anti-S100a10 (1:200, PA5-95505, Invitrogen), and anti-beta III tubulin (1:400, ab78078, Abcam). After washing with PBS, the sections were incubated with appropriate secondary antibodies at 37°C for 2 h and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) the next day. Fluorescence images were acquired with a fluorescence microscope (BX51, Olympus). Three non-consecutive sections (imaged using a 20× objective lens) from each subject and five separate visual fields from each section were randomly selected and analyzed. All images were analyzed using ImageJ (NIH), and all analyses were performed in a blinded manner.

Protein was isolated from spinal cord tissue or cultured cells by homogenizing the samples using RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher). The protein concentration was determined by the BCA assay, and 20–30 μg of protein from each sample was separated by SDS-PAGE (10 or 12%) and transferred onto NC membranes. After blocking with 5% BSA for 1 h at room temperature, the membranes were incubated with the following specific primary antibodies overnight at 4°C: C3 (ab200999, Abcam, 1:1000), S100a10 (PA5-95505, Invitrogen, 1:1000), NF-κB (8242, Cell Signaling Technology, 1:1000), pNF-κB (3033, Cell Signaling Technology, 1:1000), Notch1 (3608, Cell Signaling Technology, 1:1000), JAK2 (3230, Cell Signaling Technology, 1:1000), pJAK2 (AF3022, Affinity, 1:1000), STAT3 (12640, Cell Signaling Technology, 1:1000), pSTAT3 (ab76315, Abcam, 1:1000), PI3K (60225-1-Ig, Proteintech, 1:1000), pPI3K (AF3241, Affinity, 1:1000), Akt (4691, Cell Signaling Technology, 1:1000), pAkt (4060, Cell Signaling Technology, 1:1000), and β-actin (66009-1-Ig, Proteintech, 1:1000). After washing with TBST, the membranes were incubated with corresponding secondary antibodies for 1 h and then developed with ECL-Plus Reagent (Millipore), and the bands were visualized using an Amersham Imager 600 (General Electric).