Bx63 epifluorescence microscope

CoroNa^TM Green AM (Life Technologies), which is a Na⁺-specific fluorescent dye, was used to detect the accumulation of Na⁺ in sweet potato root cells^{5 (link)}. The roots were collected after NaCl treatment and placed in a centrifuge tube containing fresh incubation solution (200 mM NaCl, 20 μM CoroNa^TM Green AM (Life Technologies), and 0.02% pluronic acid) for 2 h. Afterward, they were washed several times with distilled water. An Olympus BX63 epifluorescence microscope was subsequently used to visualize intracellular Na⁺ accumulation (as indicated by the green fluorescence). All images were taken using the same settings and exposure times to allow direct comparisons. Image-Pro Plus 6.0 software was used to quantify the relative fluorescence intensity in the different root zones.

Immunofluorescence was performed on fresh frozen acetone-fixed 5 µm sections of OCT-embedded lung tissue. All antibodies were from Thermo Fisher Scientific, unless otherwise stated. To detect Shh: goat anti-Shh clone N19 (Santa Cruz Biotechnology, Dallas, TX, USA), followed by donkey anti-goat biotin (Alpha Diagnostic International, San Antonio, TX, USA) and streptavidin-Alexa Fluor 555 or anti-biotin Alexa Fluor 488; E-cadherin: anti-E-cadherin followed by anti-rat IgG1 PE; SiglecF: anti-mouse SiglecF and anti-rat IgG2a eFluor570; CD16: anti-mouse CD16/32 followed by anti-rat IgG2a eFluor570; CD45: anti-mouse CD45.2-FITC; and CD31: anti-mouse CD31-APC. Data were captured on a BX63 epifluorescence microscope (Olympus, Tokyo, Japan) or an LSM 710 confocal microscope (Zeiss, Jena, Germany) and analyzed using cellSens (Olympus) and Image J (National Institutes of Health, Bethesda, MD, USA) software. Magnifications are given as power of microscope objective; where multiple magnifications are used in a single figure, scale bars are also included.

Fluo-4 AM (Molecular Probes) and ION Potassium Green-2 AM (APG-2 AM, Abcam) are used to detect the concentration of Ca²⁺and K⁺, respectively, according to the published methods^{108 (link),109 (link)}. For Ca²⁺ measurements, the treated C. elegans were loaded with 100 µM Fluo-4 AM for 1 h in M9 buffer. C. elegans were washed >3 times and then photographed by Olympus BX63 epifluorescence microscope. For K⁺ measurements, treated C. elegans were labeled in 5 μM APG-2 AM in each buffer of different potassium concentrations for 1 h. The labeled C. elegans were cultured in the corresponding buffer with added OP50 for 1 h to remove the free dye in the intestine. The washed C. elegans were photographed under the same conditions. The excitation and emission wavelengths of Fluo-4 AM were 489 and 508 nm. For APG-2 AM, they are 526 and 550 nm, respectively. At least three repeats were performed for each condition and at least 20 animals were measured per treatment. The average optical density of each worm was measured using Image Pro Plus v6.0.

Analysis of subcellular localization of drugs was performed with a multispectral Leica (Wetzlar, Germany) TCS SP8 confocal microscope operating with 405 nm (argon–UV) and WLL2 (White Light Laser) laser lines. ƴ-H2AX indirect immunofluorescence was visualized with an Olympus (Tokyo, Japan) BX63 epifluorescence microscope equipped with a CoolLED’s pE-300 light source and Olympus DP74 camera. During all the experiments, cell growth and morphology were monitored with and Leica DMIL LED inverted microscope equipped with a Leica DFC 420c camera. In addition, time-lapse video microscopy of living cells after combined treatment was carried out with a Leica DMI6000B microscope with an incubation system and an OrcaR2 monochrome digital camera for image detection (Hamamatsu Photonics, Shizuoka, Japan).

For immunofluorescence experiments, cells were grown on 12 mm round glass coverslips until ~80% confluency and fixed in 2% MeOH (15 min on ice). Coverslips were washed three times for 5 min in PBS, permeabilized for 15 min (0.5% Triton X-100 in TBS), blocked for 30 min (5% BSA in TBST), and incubated with primary antibody against NHE1 (antibody details see below) in TBST + 1% BSA at RT for 1.5 h. Coverslips were again washed in TBST + 1% BSA, and incubated with AlexaFluor568 conjugated secondary antibody (1:600 in TBS + 1% BSA) for 1.5 h. Finally, coverslips were incubated with DAPI (1:1000) for 5 min to stain nuclei, washed in TBST, and mounted in N-propyl-galleate mounting medium (2% w/v in PBS/glycerol). Cells were visualized using the ×60/1.35 NA objective of an Olympus Bx63 epifluorescence microscope. Image adjustments were carried out using ImageJ software. Line scans were performed using the ColorProfiler ImageJ software plugin.

Tetramethyl rhodamine ethyl ester (TMRE) ((MCE, # 115532-52-0)) is a lipophilic cation to detect the membrane potential (ΔΨm) of the mitochondrial^{105 (link)}. The uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) a protonophore disrupt ATP synthesis due to the collapse of H+ gradient, resulting in a decrease in mitochondrial membrane potential Synchronized N2 worms were treated according to the liquid assay. After being treated by respective bacteria and compounds, worms were resuspended in M9 containing 8 μM TMRE. After 2 h treatment, worms were washed with M9 for 3 times to remove dye thoroughly. Worms were photographed using Olympus BX63 epifluorescence microscope at ×10 magnification with excitation at 550 nm and emission at 575 nm. To quantify the mitochondrial membrane potential, we converted the fluorescence intensity of each worm to the optical density value and used the cumulative optical density/ worm area as the quantitative value of membrane potential intensity. At least three repeats were performed for each condition and at least 20 animals were measured per treatment. The average optical density of each worm was measured using Image Pro Plus v6.0.