Sigma cocktail

Proteins extracted from ipsilateral brain tissue of mouse and cell lysates with radioimmunoprecipitation assay (RIPA) lysis buffer (1% Triton X‐100, 50 mmol/L Tris–HCl, pH 7.5, 100 mmol/L NaCl) containing protease inhibitors and phosphatase inhibitors (Sigma cocktail, Sigma‐Aldrich, St. Louis, MO, USA) were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose filter. Membranes were blocked for 1 h in 5% dried skimmed milk and incubated with the following primary antibodies overnight at 4°C: anti‐P62 (PM045, MBL, Japan), anti‐LAMP2 (sc‐18822), anti‐LC3B (M186‐3, MBL), anti‐Bax (2772, Cell Signaling Technology), anti‐Bcl‐2 (2870, Cell Signaling Technology), and anti‐GAPDH (sc‐3650062). The blots were incubated with horseradish peroxidase‐conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h and visualized using an enhanced luminescence kit (Millipore).

The forward fontanelle, cut at a coronal slice thickness of 2 mm from the optic chiasma, was harvested 3 or 14 days after reperfusion. Samples were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1% Triton X-100) containing protease inhibitors (aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and pepstatin) and phosphatase inhibitors (Sigma cocktail, Sigma-Aldrich, St. Louis, MO, USA). For each sample, 100 μg total protein was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by electrophoretic transfer to polyvinylidene difluoride membranes. Membranes were incubated overnight at 4°C in a 1 : 1000 dilution of primary antibodies against caspase-3 (Abcam, Cambridge, UK), synaptophysin (Abcam), MAP-2 (Cell Signaling Technology, Boston, MA, USA), MBP (Abcam), brain derived neurotrophic factor (BDNF) (Abcam), or basic fibroblast growth factor (b-FGF) (Abcam). Chemiluminescent detection of antigens was performed following incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min at room temperature using an enhanced luminescence kit (Millipore, Billerica, MA, USA).

Ipsilateral cerebral tissue was homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors (aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and pepstatin) and phosphatase inhibitors (Sigma cocktail; Sigma Aldrich, St. Louis, MO, USA). After sonication, protein concentration was determined and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was then performed. Nitrocellulose membranes were incubated with anti-arginase-1(Arg1) (1:500; 9819, Cell Signaling Technology, Boston, MA, USA) and β-actin (1:1000; 1616, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4 °C overnight, followed by corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology Inc.). Finally, immunoblots were visualized using an enhanced luminescence kit (Millipore, Billerica, MA, USA). Blot intensities were then semi-quantified using Image J software (National Institute of Health), with β-actin as an internal control. Blot intensities were also examined in a blinded manner.