Cm 1000 cryostat

Segments of or aortas (chronic treatment) and carotid arteries (ex vivo treatment) were frozen in OCT compound (Tissue-Tek) and sliced into sections (10 μm) using a Leica CM 1000 cryostat. The arterial sections and treated HUVECs were incubated in DHE- (5 μM; Invitrogen) containing normal physiological saline solution (NPSS) at 37°C for 15 min. NPSS contained (mM): 140 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 5 HEPES (pH 7.4). Fluorescence images were obtained with the Leica TCS SP8 Confocal Laser Scanning Microscope System (Leica Microsystems, Germany) by measuring the fluorescence intensity at excitation 515 nm and emission 585 nm.

The intracellular ROS levels in HUVECs and cross-sectional areas of aortas were detected by DHE probe as in our previous study [19 (link)]. Segments of mouse thoracic aortas (ex vivo treatment or in vivo treatment) and carotid arteries (ex vivo treatment) were embedded in OCT compound (Tissue-Tek, Sakura, Nagano, Japan) and cut into sections of 10 μm thickness using a Leica CM 1000 cryostat at −20 °C. Frozen sections of aortas and carotid arteries or cultured HUVECs were washed with PBS and incubated for 15 min with DHE (5 μM)-containing normal physiological saline solution (NPSS) at 37 °C in the dark. After the samples were washed with PBS to remove the dye, fluorescence images were observed by the Leica TCS SP8 Confocal Laser Scanning Microscope System (Leica Microsystems, Germany; 515 nm excitation; 585 nm emission). DHE fluorescence intensity was measured by ImageJ 1.53a software.