Western Blot Analysis of Brain Tissue

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.