Rabbit anti ym1

Immunohistochemistry was performed on 60 μm coronal sections and standard immunostaining techniques were employed. Sections were incubated with rabbit anti-Ym1 (1:600, Stemcell Technologies Inc., Vancouver, BC), washed in 1 × PBS (3 times) and incubated with biotinylated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA) for 2 hours at room temperature. Sections were incubated in avidin-biotin-horseradish peroxidase solution (Vectastain elite ABC kit, Vector Laboratories) for 1 hour and then reacted with 3,3′-diaminobenzidine (Vector Laboratories) for color development and mounted for immunohistochemical analysis using a Leica DM4000B microscope (Leica Microsystems, Exton, PA, USA).

Proteins from ipsilateral cortical tissue were extracted using RIPA buffer, equalized, and loaded onto 5–20% gradient gels for SDS PAGE (Bio-Rad; Hercules, CA). Proteins were transferred onto nitrocellulose membranes, and then blocked overnight in 5 % milk in 1 × TBS containing 0.05 % Tween-20 (PBS-T). The membrane was incubated in mouse anti-arginase 1 (N-20) (1:1000; BD Transduction Laboratories), rabbit anti-Ym1 (1:1000; Stem Cell Technologies, Vancouver, BC), or rabbit anti-GAPDH (1:2000; Sigma) overnight at 4°C, then washed three times in TBS-T, and incubated in appropriate HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h at room temperature. Membranes were washed three times in TBS-T, and proteins were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL). Chemiluminescence was captured ChemiDoc™ XRS+ System (Bio-Rad), and protein bands were quantified by densitometric analysis using BioRad Molecular Imaging Software. The data presented reflects the intensity of target protein band normalized based on the intensity of the endogenous control for each sample (expressed in arbitrary units).