Arginase 1 arg 1

PFA fixed brain sections or cells were washed three times with PBS for 5 minutes, incubated with 0.3% TritonX-100 for 10 minutes and blocked with 5% bovine serum albumin for 60 minutes at RT, then incubated with primary antibodies at 4 °C overnight. The primary antibodies are used as follows: rat PDGFR-α (1:100; Santa Cruz, CA), mouse PDGFR-α (1:100; eBioscience, CA), NG2 (1:200; Millipore, CA), MBP (1:200; Abcam, Cambridge, UK), SMI32 (1:200; Millipore), adenomatous polyposis coli (APC, 1:200; Millipore), Ki67 (1:200; Millipore/ 1:200, Servicebio, Wuhan, CN), glial fibrillary acidic protein (GFAP, 1:500, Millipore), NeuN (1:200; Millipore), Iba-1 (1:200; WAKO, Osaka, Japan), CD206 (1:200; R&D system, CA), Arginase-1 (Arg-1, 1:50; Santa Cruz), CD31 (1:200; Abcam). After rinsing thrice with PBS, the samples were incubated with corresponding secondary antibodies for 60 minutes at RT. Nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI, 1:1000; Life Technologies, Mulgrave, VIC, AUS).

Mouse brains were obtained under deep anesthesia 1 and 3 days after surgery, and the ipsilateral hemisphere was used for Western blot analysis. For cells and brain tissue, the lysates were prepared using RIPA lysis buffer (Millipore) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The Western blot protocol was performed as previously described [21 (link)]. The primary antibodies used were iNOS (1:500; Abcam), Arginase-1(Arg-1; 1:500; Santa Cruz), CD206 (1:800, Abcam), LC3B (1:1000, Sigma-Aldrich), LAMP2 (1:500; Millipore), phospho-mTOR (Ser 2448) (p-mTOR; 1:500; Cell Signaling Technology, Beverly, MA, USA), mTOR (1:1000, Cell Signaling Technology), FLAG (1:1000; Abcam), caspase3 (cas3; 1:500, Cell Signaling Technology), BCL2-associated X protein (bax; 1:1000; Abcam), B cell lymphoma 2 (bcl2;1:500; Cell Signaling Technology), GAPDH (1:1000; Santa Cruz), and β-actin (1:1000; Santa Cruz). The immunoblots were detected using an enhanced chemiluminescence kit (FD Technology, Shanghai, China) and obtained using an imaging system (Bio-Rad, Hercules, CA, USA) and then analyzed by ImageJ software.

After the vessels were embedded in paraffin blocks, 4-micron sections were cut using a microtome and mounted on microscope slides. After washing with 1% PBS, slides were incubated overnight at 4°C with the primary antibody: anti-CD68 (Thermo Scientific, Long Beach, NY, USA), inducible nitric oxide synthase (iNOS; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for M1-macrophages, or arginase-1 (Arg-1; Santa Cruz Biotechnology) for M2-macrophages. Slides were then washed in 1% PBS and incubated for 1 h in a dark room with the secondary antibody: fluorescein isothiocyanate–conjugated donkey anti-mouse IgG, phycoerythrin–conjugated goat anti-rabbit IgG, or Texas Red–conjugated goat anti-goat IgG (Santa Cruz Biotechnology). After slides were washed in 1% PBS for 10 min, nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (ImmunoBioScience, Mukilteo, WA, USA). Slides were then mounted with Fluoroshield, and analyzed using confocal microscopy (LSM700 system; Carl Zeiss Inc, Jena, Germany).

Protein extracts from different groups of cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis before transferred to polyvinylidene fluoride (PVDF) membranes, and then incubated with the corresponding primary Abs at 4°C overnight after blocking with 5% skim milk. The following primary Abs were used: monoclonal Ab against GAPDH (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), SIRT3 (1:600; Cell Signaling Technology), nuclear factor (NF)-κB (1:600; Santa Cruz Biotechnology), interferon-γ (IFN-γ; 1:600; Santa Cruz Biotechnology), arginase-1 (Arg1; 1:600; Santa Cruz Biotechnology), inducible nitric oxide synthase (iNOS; 1:600; Santa Cruz Biotechnology), chitinase 3-like 3 (Ym1; 1:600; Santa Cruz Biotechnology), and Iba-1 (1:600; Santa Cruz Biotechnology). The sample were incubated with secondary Abs after incubated with the primary Abs. Subsequently, the membranes were washed and visualized using an electrochemiluminescence system. Protein band densities were analyzed using UN SCAN-IT gel software. β-actin was used as a loading control.