Ti s fluorescence microscope

Vero cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) (Solarbio) followed by incubating with blocking buffer (0.2% skim milk, 2% FBS, 0.1 M glycine, 1% bovine albumin, and 0.01% triton-X 100 in PBS) for 30 min after incubating with PEDV N protein monoclonal antibody (1:500) for 1 h, followed by the incubation with fluorescence-conjugated goat anti-mouse IgG (1:1000) (Zhongshan, Beijing, China) and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Solarbio) for 30 min. Fluorescence signals were captured with a Ti-S fluorescence microscope (Nikon, Tokyo, Japan). The percentage of viral infected cells was calculated by counting the cells with PEDV N-positive signal and dividing it by the number of DAPI-positive [10 (link)]. For each condition, we chose at least three views with more than 300 DAPI-positive cells and triplicated them to get the standard deviation.

SB cells grown in vitro were first smeared onto Fisherbrand Colorfrost microscope slides and fixed in −20°C MeOH/CH₃COOH (3∶1) for 1 hour at RT. The tissue section slides were prepared by Charles River Laboratories. All of the slides were washed in 2X SSC, pretreated with pepsin at 37°C, fixed with 1% formaldehyde in 0.5% MgCl₂/PBS, and dehydrated with serial concentrations of ethanol. Fluorescence human Y chromosome probes (Empire Genomics) were loaded onto the slides and denatured at 80°C. The slides were then incubated overnight at 37°C and washed with 0.4X SSC/0.3% IGEPAL at 37°C and 2X SSC/0.1% IGEPAL at RT for 2 minutes each. The slides were then counterstained with DAPI (Vector Vectashield) and observed under a Nikon Ti-S fluorescence microscope.

The cellular uptake and distribution of SLN formulations were characterized by fluorescence microscopy and flow cytometry. Briefly, B16F10 cells were seeded at a density of 2 × 10⁵ cells per well in 12-well culture plates and further cultured overnight. For imaging, coumarin-6 was incorporated into the SLNs to obtain the fluorescence-labeled composite nanoparticles. The coumarin-6 labeled formulation and free coumarin-6 at different concentrations were added to the cells without serum, and the cellular uptake was observed in a time- or dose-dependent manner. Following incubation at 37 °C, the B16F10 cells were washed twice with PBS, harvested, and analyzed using a Ti-S fluorescence microscope (Nikon, Tokyo, Japan) and a flow cytometer (Navios, Beckman, CA).

hiPSC-CMs were replated onto Matrigel-coated glass slides and maintained in CM maintenance medium. At 3 weeks, the cells were transferred into a perfusion-stimulation-chamber and incubated with 9 μM Indo-1 acetoxymethyl ester (Thermo) for 15 min at room temperature. After mounting the perfusion-stimulation-chamber on an inverted Nikon Ti-S fluorescence microscope, the cells were washed with Tyrode's solution (140 mM NaCl, 5.8 mM KCl, 0.5 mM KH₂PO₄, 0.4 mM Na₂HPO₄, 0,9 mM MgSO₄, 10 mM HEPES, 11.1 mM Glucose, 2 mM CaCl₂, pH 7.3) in order to minimize background fluorescence. Next, intracellular Ca²⁺ transients were evoked via field-stimulation at a frequency of 0.5 Hz. The Ca²⁺ transients were recorded as the Indo-1 fluorescence emission ratio F405 nm/F495 nm using an IonOptix (Milton, MA) detection system (excitation: 344 nm). At least 10 cell clusters per glass slide were measured under basal conditions as well as during superfusion with isoprenaline (1 μM). For each cell cluster, ten intracellular Ca²⁺ transients were averaged using the IonWizard analysis program to determine changes in peak amplitude and transient decay kinetics.

Cells were seeded at a density of 1×10⁵ cells/well in a 24-well plate and cultured under 5% CO₂ at 37°C for 24 h. Following 6 h transfection, changes in the rate of cell migration were measured using a wound healing assay. Briefly, cells in each well were separated by a scratch-wound, a standardized scratch made with a P-20 pipette tip, and cells were observed every 12 h for 48 h using a Nikon Ti-S fluorescence microscope. The data were analyzed using the Image-Pro Plus software. http://dx.doi.org/10.17504/protocols.io.iascaee [PROTOCOL DOI]