Anti arg 1

In immunostaining analysis, non-specific binding was blocked with 3% bovine serum (Serotec, UK), Such binding underwent permeabilization for 30mins with 0.1% Triton X-100 in 1% BSA-PBS. The sections underwent the incubation overnight at 4°C with monoclonal rabbit anti-TH (1:500; Millipore), anti-GFP (1:500; Abmart), anti-iNOS (1:500; Proteintech), anti-Arg-1 (1:200; BD), anti-Nestin (1:100; eBioscience). Subsequently, they underwent the incubation with the relevant Alexa Fluor 488-conjugated secondary anti-body (1:1000; Invitrogen) or Alexa Fluor 555-conjugated secondary antibody (1:1000; Invitrogen) for 2hrs at ambient temperature. The nucleus was stained by DAPI (Invitrogen). The control section ran under the same protocol, but the major antibodies were omitted.

After rapid brain removal, the tissue was separated from the brain and homogenized on ice with a micro-content electric tissue homogenizer (KIMBLEKONTES, USA) in frozen lysis buffer (1×PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, RIPA) containing protease inhibitors. Lysates underwent the centrifugation at 4°C for 20 min at 10,000 × g, and the authors collected the supernatants. With the use of a Bradford protein assay, the concentrations of protein were ascertained. Protein with equal amounts (30 µg) underwent the separation using the SDS-PAGE and electroblotted on nitrocellulose filter (NC) membrane (Millipore). After non-specific antibody binding was blocked with 5% non-fat dry milk, membranes underwent the incubation at 4°C overnight with monoclonal rabbit anti-TH (1:2000; Millipore), anti-iNOS (1:1000; Proteintech), anti-Arg-1(1:1000; BD). After being washed in TBST, the immunoblots underwent the incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 1 h. Using enhanced chemiluminescence (ECL) reagents (Millipore, USA), the immunoblots were developed, and they underwent the measurement using Quantity Software (Bio-Rad, CA). For the comparison of protein loading, antibody directed against GAPDH was employed.