Ultra du 897u

SIM analysis of brain sections was performed on a Nikon SIM system. Tissues were imaged at laser excitation of 488 (for A11), 561 (for CD68) nm with a 3D-SIM acquisition protocol. Sixteen-bit images sized 1024×1024 pixels with a single pixel of 0.030 µm were acquired in a gray-level range of 0–16000 to exploit the linear range of the camera (iXon ultra DU-897U, Andor) and to avoid saturation. Three-dimensional Z-stacks were scanned with a 0.125 µm step size over 2–3 µm. Raw and reconstructed images were validated with the SIM-check plugin of ImageJ [59 (link)] and only those providing satisfactory image diagnosis were included in the study. Images were finally elaborated with GIMP (Gnu Image Manipulation Program).

Glass coverslips (TH. Geyer, 25 mm diameter, No. 1.5H) were coated with poly-d-lysine solution (Sigma-Aldrich, #P6407) at a concentration of 0.1 mg ml⁻¹ for 5 min at room temperature and washed three times with PBS. Cells were seeded on poly-d-lysine-coated coverslips and live-cell microscopy was performed 12–16 h after seeding. Live-cell imaging of AP2-eGFP was performed with an inverted spinning-disk confocal microscope (PerkinElmer), with a 60× (1.42 numerical aperture, Apo TIRF, Nikon) or 100× (1.4 numerical aperture, Plan Apo VC, Nikon) oil immersion objective and a CMOS camera (Hamamatsu Ocra Flash 4). An environment control chamber was attached to the microscope to keep 37 °C and 5% CO₂. The 10-min-long movies of representative cells were taken with one frame every 3 s. Live-cell imaging of AP2-eGFP together with CLCa-tdtomato was performed with an inverted Ti microscope (Nikon) with objective TIRF illumination, with a 60× (1.49 numerical aperture, Apo TIRF, Nikon) oil immersion objective and EMCCD camera (Andor iXon Ultra DU-897U). An on-stage incubation chamber was used to keep 37 °C and 5% CO₂. The 10-min-long movies of representative cells were taken with one frame every 3 s.

The SIM on brain sections was done with a Nikon SIM system with a 100 × 1.49 NA oil immersion objective, managed by NIS elements software. Tissues were imaged at laser excitation of 488 (for Tmem119) and 640 nm (for Iba1) with a 3D-SIM acquisition protocol. Fourteen-bit images sized 1024 × 1024 pixels, with a single-pixel of 0.030 μm, were acquired in a gray level range of 0–4,000 to exploit the linear range of the camera (iXon ultra DU-897U, Andor) and to avoid saturation. Raw and reconstructed images were verified by the SIMcheck ImageJ plugin (Ball et al., 2015 (link)).