2.5. Double immunofluorescence staining and TUNEL staining

Rats were sacrificed at 24 hours after SAH induction. A series of 8 μm‐thick frozen brain tissue slices were prepared. Double immunofluorescence staining was performed as previously described.²⁸, ²⁹ The primary antibodies used were anti‐GFAP (1:500, ab53554), anti‐NeuN (1:500, ab177487), and anti‐IBA 1 (1:500, ab107159) from Abcam; anti‐Osteopontin (1:100, sc‐21742) from Santa Cruz Biotechnology Inc; anti‐Beclin 1 (1:100, NB500‐249), and anti‐Caspase‐3 (1:50, NB100‐56708) from Novus Biologicals. Corresponding secondary antibodies were purchased from Jackson Immunoresearch and were applied at a concentration of 1:500. Nuclei were counterstained with DAPI (blue). After staining, the sections were visualized and photographed with a fluorescence microscope (Leica Microsystems).

TUNEL staining was performed with an in situ cell death detection kit (TUNEL) following the manufacturer's instructions (11684795910, from Roche Diagnostics). Nuclei were counterstained with DAPI (blue). Three rat brains per group (six sections per brain) were used for quantification analysis. Images of TUNEL‐positive cells in designated locations were captured at ×200 magnification by an independent observer with a fluorescence microscope (Leica Microsystems). Image J software (Image J 1.4, NIH) was used for cell counting. Also the data were presented as the average number of TUNEL‐positive cells per square millimeter (cell/mm²).