Sp8 mp confocal laser scanning microscope

Brains were perfusion‐fixed and frozen OCT‐embedded sagittal sections (35 μm) were cut. Sections were immunolabeled for IBA‐1 (rabbit anti‐mouse Iba‐I antibody; 1:200, Fijifilm; overnight at 4°C) and the endothelial marker endomucin (rat monoclonal anti‐mouse endomucin antibody; 1:50, Invitrogen; overnight at 4°C) to identify microglia and capillary endothelial cells in the brain, respectively. Confocal images were obtained using Leica SP8 MP confocal laser scanning microscope. The relative staining intensity for IBA‐1 positive perivascular microglia per region of interest was assessed.

Brains collected after perfusion with ice-cold PBS were fixed in 4% PFA for 24 h and subsequently cryoprotected through immersion in 30% sucrose, followed by embedding in OCT medium. Frozen brain OCT blocks were cryosectioned at a thickness of 35 µm, and the sections were stored in cryoprotectant solution at −20 °C until staining. The free-floating sections were immunolabeled using an anti-endomucin primary antibody (1:75, EMD Millipore, Burlington, MA, MAB2624) and an anti-rat Alexa 488 secondary antibody (1:500, Invitrogen, Molecular Probes, Carlsbad, CA, USA) to identify capillary endothelial cells in the mouse brain. Nuclear counterstaining was performed using DAPI (Sigma). Confocal images were acquired using a Leica SP8 MP confocal laser scanning microscope. Senescent endothelial cells were identified in 20× images by co-localizing RFP (p16+ senescent cells) with Alexa 488 green fluorescence signal in the hippocampal region. Double-positive cells were quantified using the Image J count plugin and expressed as the average number of senescent endothelial cells per field in each group.