Stedycon microscope

MC3T3 E1 cells on a coverslip in a 12-well plate supplemented with Opti-MEM were incubated with P4-FITC or P4-FITC@Si in a humidified atmosphere containing 5% CO₂ at 37 °C for 4 h. The cells were washed 3 times with PBS and fixed with 4% paraformaldehyde at 37 °C for 30 min. After washing the cells three times with PBS, the cells were immersed in PBS containing CellTracker Red CMTPX (Thermo Fisher Scientific Korea Ltd. (Seoul, Republic of Korea) (5000:1 dilution) for 30 min to stain the cytoplasm. The stained cells on the cover slip were washed 3 times with PBS and applied on the mounting drop with DAPI on a slide glass. The confocal images were taken along the XY plane and z-axis using a STEDYCON microscope (abberior instruments GmbH, Göttingen, Germany) with a 100× oil immersion objective lens. MC3T3 E1 cells show rhodamine-labeled cytoplasm (red), FITC-labeled p4 peptide or P4-silica particles (green), and DAPI-labeled nucleic acids (cyan hot). Laser power was set at 10%, and the pixel size was 30 nm. Z-stacks of the cells were acquired with a 100 nm step size. ImageJ software was used to generate videos of the z-stacks and 3D reconstructions of the confocal images.

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.