Filter set 20 fluorescing fluorophore

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)).