Axiovision

Briefly, fluorescence microscopy was done on an Observer A1 inverted microscope (Zeiss, Germany) using a 25 × 0.8 numerical aperture water immersion objective and a 175 W Xenon lamp as a light source. Before imaging, DRG neurons were incubated with 4 μM Fura-2 at 37 °C for 30–40 min and washed with extracellular solution at 37 °C for another 30 min. Excitation light was passed either through a 340-BP 30 filter or a 387-BP 16 filter. Two filters were switched by an ultra-high speed wavelength switcher Lambda DG-4 (Sutter, Novato, CA). Emissions elicited from both excitation wavelengths were passed through a 510-BP 90 filter and collected by a charge-coupled device camera (Zeiss). Different solutions were applied by multi barrel perfusion system (WAS02, DITEL, Prague). AxioVision software (Zeiss) was used to record image data. After background subtraction in each channel (B₃₄₀, B₃₈₀), the ratio (R) of fluorescence elicited by two excitation light, B₃₄₀ and B₃₈₀, was calculated: 59 (link). Data weres analysed by using AxioVision and Matlab (MathWorks, Natick, Massachusetts).

Calcium imaging was performed as previously described (Chen et al., 2014 (link)). Fluorescence microscopy was done on an Observer A1 inverted microscope (Zeiss, Germany) using a 25 × 0.8 numerical aperture water immersion objective and a 175W Xenon lamp as a light source. Before imaging, DRG neurons were incubated with 4 mM Fura-2 at 37°C for 40 min and washed with extracellular solution at 37°C for another 30 min. Excitation light was passed either through a 340 BP 30 filter or a 387 BP 16 filter. Two filters were switched by an ultra-high speed wavelength switcher Lambda DG-4 (Sutter, Novato, CA). Emissions elicited from both excitation wavelengths were passed through a 510 BP 90 filter and collected by a charge-coupled device camera (Zeiss). Different solutions were applied by multi barrel perfusion system (WAS02, DITEL, Prague). AxioVision software (Zeiss) was used to record image data. After background (B₃₄₀, B₃₈₀) subtraction in each channel (F₃₄₀, F₃₈₀), the ratio (R) of fluorescence elicited by two excitation light was calculated: R=(F₃₄₀ – B₃₄₀) / (F₃₈₀ – B₃₈₀). Data was analyzed in AxioVision, Matlab (MathWorks, Natick, Massachusetts), and GraphPad prism (GraphPad Software Inc., San Diego, CA).