Plan apo vc

Immunofluorescence images and actin/keratin retrograde flow experiments were performed in a Nikon TiE inverted microscope with a spinning-disc confocal unit (CSU-WD, Yokogawa) and a Zyla scientific complementary metal–oxide–semiconductor camera (Andor) with μManager (version 1.4.22), using a ×60 objective (Plan Apo; numerical aperture (NA), 1.2; water-immersion type). Epifluorescence images were taken on an automated inverted microscope (Nikon ECLIPSE Ti) using MetaMorph (NIS Elements) imaging software (version 7.7.10) and a ×60 objective (Plan Apo VC; NA, 1.4; oil-immersion type). Higher-resolution confocal images (Extended Data Fig. 4) and three-dimensional segmentation of nuclei (Fig. 5 and Extended Data Fig. 7b–f) were acquired using a ZEISS LSM 880 inverted confocal microscope with Airyscan and a ×63 1.46-NA oil-immersion objective and ZEN (ZEISS, version 2.3 SP1 FP3 black) software. Spheroids were imaged using an inverted ZEISS LSM 880 confocal microscope with an oil-immersion ×40 objective with an NA of 1.3.

Betz cell bodies of two samples (SKO11, SKO19, left hemisphere) were microphotographed with a Nikon C2 confocal microscope, with 20 × objective (Plan Apo VC NA = 0.75) and NIS Elements AR acquisition software (version: 4.30) using 488-nm and 561-nm laser lines. Giant pyramidal cells were determined according to the characteristics listed above. Cell bodies were identified by their PV immunopositivity. We counted the terminals contacting the cells’ somata, measured their area and determined their PV, VIAAT, or vGluT1 content. A contact was confirmed when no hiatus could be observed between the cell body and terminals in the same focal plane.