Ckx53 fluorescence microscope

Cell morphology was determined using an Olympus CKX53 fluorescence microscope with a DAPI filter. The same procedure as that performed with the measurement of the non-radical H₂O₂ was carried out. All images were captured with a 10x objective and analyzed with Olympus DP-23 software (Olympus Corporation, JPN).

Before performing
the resting membrane potential assay, we performed the dose–response
activity of quinidine and compound 13a, similar to the
effective concentration assay. The only exception was that both compounds
were serially diluted in two-fold steps, from 400 to 3.12 μM
for quinidine and from 100 to 1.56 μM for compound 13a. The resting membrane potential assay was performed as previously
described.^{40 (link)} Briefly, A549 cells were treated
with 20 μM DiBAC₄(3) (Sigma) for 20 min at 37 °C
in the dark. After labeling, cells were washed and treated with either
quinidine (200 μm) as a positive control or compound 13a (25 and 50 μM) for 16 h. Labeled cells were also
left untreated as the DiBAC₄(3) control. Wide-field images
were taken with an Olympus CKX53 fluorescence microscope.

Reagents were purchased from Adamas (Basel, Switzerland), Acros (Pittsburgh, PA, USA), TCI (Tokyo, Japan), Sigma-Aldrich (St. Louis, MO, USA), and SCRC (Shanghai, China). All reagents were of analytical grade and obtained from commercial suppliers. Dichloromethane (CH₂Cl₂) was distilled over calcium hydride. Triethylamines were stored with molecular sieves (4A) before use. The water used in all experiments was purified using a Millipore Milli-Q water system (Billerica, MA, USA). ¹H NMR (400 MHz) spectra were recorded on a Bruker Ascend 400 (Fällanden, Switzerland) in CDCl₃ as a solvent with TMS as the internal standard. UV–vis absorption spectra were recorded on a Shimadzu UV-1800 spectrometer (Kyoto, Japan) in 1.0 cm path-length quartz. Fluorescence spectra were recorded on an Agilent Cary eclipse fluorometer (Santa Clara, CA, USA) with both excitation and emission slits set at 5 nm. The pH value measurements were carried out with a Mettler Toledo Delta 320 pH meter (Zurich, Switzerland). The fluorescence microscopy images of plant tissue were recorded with an Olympus CKX53 fluorescence microscope (Tokyo, Japan). Whole-plant real-time images were recorded with ImagingPhotonIMAGER (Paris, France). All experiments were performed in flame-dried glassware using dry solvents unless otherwise noted.

The paraffin-embedded sections were dewaxed, rehydrated, and followed by standard IHC protocol [14 (link)]. Sections were incubated with primary antibodies (Table 2) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibodies for 30 min. Immunoreactivity was developed using a DAB substrate kit (Maixin biotech company, Fuzhou, China). Nuclei were counterstained with hematoxylin. Immunofluorescent staining was performed using a similar protocol, followed by incubation with Cy3 or DyLight 488-conjugated secondary antibodies. All the slides were mounted with an anti-fade mount and visualized with an Olympus CKX53 fluorescence microscope (Tokyo, Japan).

Cells were photographed using Olympus CKX53 fluorescence microscope at 72 h post-DENV-2 infection and then collected by centrifuging at 300 × g for 10 min. The collected cells were fixed with 4% paraformaldehyde (PFA) before being analyzed by flow cytometer CytoFLEX. GFP was a reporter gene carried by EBV virus in Akata EBV + cells.

Reprogrammed cells were gently washed with DPBS and fixed in 4% paraformaldehyde at room temperature for 20 min. The cells were washed again with 0.05% Tween-20 (Sigma-Aldrich) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) at room temperature for 15 min. After washing, they were blocked with 4% Donkey Serum at 4 °C overnight and wrapped in parafilm. Primary antibodies, SSEA4, OCT4, SOX2, TRA-1-60, and NANOG, were added to the cells and then, incubated at 4 °C overnight. The secondary antibodies, Alexa Fluor 594 anti-rabbit or Alexa Fluor 488 anti-rabbit antibodies (Life Technologies, Eugene, OR, USA), were supplemented after washing with 0.05% Tween-20. The samples were incubated at 4 °C overnight in the dark and stained with UltraCruz Aqueous Mounting Medium with DAPI (Santa Cruz Biotechnology, Dallas, TX, USA) for observation.
A CKX53 fluorescence microscope (Olympus, Tokyo, Japan) with a U-RFL-T fluorescence lamp (Olympus) was used to visualize the cells. Image analysis and colocalization studies were carried out using the Ocular Image Acquisition Software, version 2.0.1.496 (Digital Optics Limited, Auckland, New Zealand) [26 (link)].