Axio examiner z1 microscope

A fixed-stage Zeiss Axio Examiner Z.1 microscope (Carl Zeiss Microscopy GmbH, Germany) equipped with a confocal scanning unit (Yokogawa CSU-X1; Yokogawa Electric Corporation, Japan) was used for all microscopic analyses. Confocal images were captured using a high-speed digital camera (CCD C9300-221; Hamamatsu Photonics K.K., Hamamatsu City, Japan).

Diaphragm-phrenic nerve preparations were maintained ex vivo in Liley’s solution gassed with 95% O₂, 5% CO₂ at room temperature [21 (link)]. The recording chamber had a volume of approximately 1 mL and was perfused at a rate of 1 mL/min. The nerve was drawn up into a suction electrode for stimulation with pulses of 0.1 ms duration. The preparation was placed on the stage of a Zeiss Axio Examiner Z1 microscope (Carl Zeiss MicroImaging, Göttingen, Germany) fitted with incident light fluorescence illumination with filters for 547 nm/red (Zeiss filter set 20) fluorescing fluorophore (Carl Zeiss MicroImaging). At the beginning of the experiment, the compound muscle action potential (CMAP) was recorded using a micropipette with a tip diameter of approximately 10 µm filled with bathing solution. The electrode was positioned so that the latency of the major negative peak was minimized. The electrode was then positioned 100 µm above the surface of the muscle, and CMAP was recorded.
For recordings in the presence of tubocurarine, the chamber was filled with 2 mL (300 nM, 500 nM, 800 nM, or 1000 nM) of d-tubocurarine chloride (Sigma Aldrich Chemie, München, Germany). During the curare treatment, trains of 25 repetitive nerve stimulations (5 Hz) were performed at 2 min intervals, and the ratio of CMAP amplitudes (mean (20th–25th)/2nd) was calculated [22 (link)].

Diaphragm-phrenic nerve preparations were maintained ex vivo in Liley's solution gassed with 95% O₂, 5% CO₂ at room temperature (55 (link)). The recording chamber had a volume of approximately 1 ml and was perfused at a rate of 1 ml/min. The nerve was drawn up into a suction electrode for stimulation with pulses of 0.1 ms duration. The preparation was placed on the stage of a Zeiss Axio Examiner Z1 microscope (Carl Zeiss MicroImaging) fitted with incident light fluorescence illumination with filters for 547 nm/red (Zeiss filter set 20) fluorescing fluorophore (Carl Zeiss MicroImaging). At the beginning of the experiment, the compound muscle action potential (CMAP) was recorded using a micropipette with a tip diameter of approximately 10 μm filled with bathing solution. The electrode was positioned so that the latency of the major negative peak was minimized. The electrode was then positioned 100 μm above the surface of the muscle, and CMAP was recorded. For recordings in the presence of d-tubocurarine, the chamber was filled with 2 ml (300, 800 or 1000 nM) of d-tubocurarine chloride (Sigma Aldrich). During the curare treatment, trains of 25 repetitive nerve stimulations (5 Hz) were performed at 2 min intervals, and the ratio of CMAP amplitudes (mean (20th–25th)/2nd) was calculated (44 (link),56 (link)).

Spheroids of H1299 shLKB1 and pLKO.1 or H157 stable cells were dyed using 1 μM of Red CellTracker (Invitrogen). The H1299 stable spheroids were imaged at 0, 6, and 21 hours post-invasion, and H157 stable spheroids were imaged at 0 and 24 hours post-invasion, using a standard upright Zeiss Axio Examiner Z1 microscope with 20x water immersion objective (1.0 NA DIC (UV) VIS-IR). The second harmonic generation (SHG) signal was obtained using a bandpass 380–430 nm cube. To image the cells stained with Red CellTracker, a bandpass of 570–610 nm cube with a long pass of 550 nm was used. Images were taken with a Coherent Chameleon Verdi laser at 790 nm wavelength. Each multiphoton microscopy image is 512 × 512 pixels in size (425 × 425 μm); each pixel is 0.83 μm. Z-stack images were taken with a 1 μm interval.