Tcs sp5 dm6000cfs

For Ca²⁺-sensitive dye loading, acute coronal VNO slices were incubated (60 min; 5°C) in circulating S₂ (storage chamber) containing Cal-520/AM (4.5 µM; Biomol) and 0.0005% pluronic F-127 (20% solution in DMSO; Thermo Fisher Scientific). After washing five times (S₂), VNO slices were transferred to a recording chamber (Luigs & Neumann) on an upright fixed-stage scanning confocal microscope (TCS SP5 DM6000CFS, Leica Microsystems) equipped with a 20×/1.0 NA water immersion objective (HCX APO L, Leica Microsystems) and infrared-optimized differential interference contrast (IR-DIC) optics. Bath solution (S₂) was continuously exchanged (∼5 ml/min; gravity flow). Cal-520 was excited using the 488-nm line of an argon laser. Changes in cytosolic Ca²⁺ were monitored over time at 1.0-Hz frame rates. Neurons were stimulated at decreasing ISIs of 180, 60, and 30 s.

In vitro imaging of VSN activity in acute coronal VNO slices was performed as described (Wong et al., 2018 (link)). Briefly, for bulk loading, slices were incubated (90 min; 5°C) in circulating S₂ with the Ca²⁺-sensitive dye CAL520/AM (4.5 μM; Biomol, Hamburg, Germany) and 0.05% Pluronic F-127 (20% solution in DMSO; Thermo Fisher Scientific, Schwerte, Germany). After washing (5×, S₂), slices were transferred to a recording chamber (Luigs & Neumann, Ratingen, Germany) mounted on an upright fixed-stage scanning confocal microscope (TCS SP5 DM6000CFS, Leica Microsystems) equipped with a 20×/1.0 NA water immersion objective (HCX APO L, Leica Microsystems), and infrared-optimized differential interference contrast optics. Slices were continuously superfused with oxygenated S₂ (~5 ml min^–1; gravity flow). CAL520 was excited at 488 nm (multi-line argon laser; <25% laser power) and fluorescence was detected within a 500–600 nm spectral band. Changes in cytosolic Ca²⁺ were monitored over time at 1.0 Hz frame rate (1024×512 pixels; 400 Hz bidirectional scanning frequency) using LAS AF software (Leica Microsystems).