Plan apochromat dic oil

Micrographs were captured with an LSM510 confocal microscope equipped with either 63 × Zeiss Plan-APOCHROMAT oil, 1.4NA or 100 × Zeiss Plan-APOCHROMAT DIC oil, 1.46NA under environmental control at 37 °C and 5% CO₂ (Zeiss, Thornwood, NY, USA).

Microvessels were plated on a glass slide coated with Cell Tak (Corning, NY) and fixed in PBS/PFA 4% for 15 min at room temperature (RT).
Brain slices or microvessels on glass slides were immersed in the blocking solution (PBS/normal goat serum (NGS) 5%/Triton X-100 0.5%) for 1 hr at RT and then incubated with primary antibodies (see the Key resources table) diluted in the blocking solution 12 hr at 4°C. After three washes in PBS, slices were incubated for 2 hr at RT (or overnight for STED experiments) with secondary antibodies and Hoechst dye, rinsed in PBS, and mounted in Fluormount G (Southern Biotech, Birmingham, AL) for confocal analysis or Abberior Mount solid antifade medium for STED imaging.
Tissues were imaged using a 40× objective on a Zeiss Axio-observer Z1 with a motorized XYZ stage (Zeiss, Oberkochen, Germany). For STED imaging, we used a STEDyCON microscope (Abberior Instruments, Göttingen, Germany) with a 100×/1.46 Plan-ApoChromat DIC Oil (Zeiss). Alexa 594 and Star-red fluorescence was depleted with a laser at 775 nm. The pixel size was set to 25 nm, with a 1.13 pinhole.