Ja31 14

The expression of Sphk1 (NOVUS Biologicals, JA31-14), MAPLC3 II (Cell Signaling Technology, D11), SQSTM1/p62 (Abcam, ab155686) and TRAF2 (Abcam, ab126758) in OGDR and MCAO model systems was measured via Western blotting. Brie y, protein levels in ischemic penumbra tissue samples were quanti ed via BCA assay. Next, 10-12% SDS-PAGE was used to separate the proteins, and they were transferred onto cellulose acetate membranes. Blots were next blocked for 2 h using 5% non-fat milk followed by overnight probing with primary antibodies at 4°C. Blots were then washed prior to probing for 1 h with appropriate uorescent secondary antibodies. Blots were then imaged with an infrared Odyssey 3.0 instrument, with β-actin being used as a means of normalizing protein expression across samples.

Immuno uorescent staining was used to visualize the microglial expression of Sphk1 (NOVUS Biologicals, JA31-14), MAPLC3 II (Cell Signaling Technology, D11) , SQSTM1/p62 (Abcam, ab155686), and TRAF2 (Abcam, ab126758) in our experimental system. Brie y, we initially used xylene to depara nize ischemic penumbra tissue sections from model animals. Sections were then treated with citric acid to facilitate antigen retrieval, followed by three washes in PBS. Samples were next blocked for 1 h using serum, followed by probing overnight at 4°C with appropriate primary antibodies, further washing, and probing for 1 h at room temperature using uorescently conjugated secondary antibodies. Samples were then imaged via Olympus FV300 confocal microscope (200×). To ensure accuracy, 4-5 elds of view in different cortical regions were selected at random, with positive areas being averaged across these regions.