Fv10 asw 3.0 viewer

For immunofluorescence, rabbit antibodies of ATXN2L (Sigma-Aldrich), E-cadherin (Cell Signaling), and vimentin (Cell Signaling), and mouse antibody G3BP1 (Abcam) were used. Immunofluorescence staining was performed as previously described^{3 (link)}. Cells were seeded onto glass-bottom dish and fixed by polyformaldehyde. Cellular membrane was permeabilized by 0.2% Triton X-100/phosphate-buffered saline (PBS) solution. After blocked by 0.5% Tween-20/PBS for 1 h, dish was incubated with antibodies against ATXN2L, E-cadherin, vimentin, or G3BP1 for 12 h. Then cells were incubated by secondary antibodies, and nucleus were stained by 4′,6-diamidino-2-phenylindole. Cells were observed by Olympus FV10i Olympus laser confocal system (Olympus, Tokyo, Japan), and analyzed by FV10-ASW 3.0 Viewer (Olympus). Staining intensity were half quantified using Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MA, USA).

SOSG (Molecular Probes Inc. Eugene, OR, USA) was applied to the U266 cells in order to visualize ¹O₂ production. Cell suspension treated with H₂O₂ was incubated with 50 μM SOSG for 30 min in dark. Following incubation, U266 cells were gently washed with 20 mM K-buffer, and consequently the ¹O₂ imaging was performed by a confocal laser scanning microscope, Fluorview 1000 (Olympus Czech Group, Prague, Czech Republic). The transition images were obtained by transmitted light detection module with 405 nm excitation with a near-ultraviolet (UV) laser diode and Nomarski DIC filters (Olympus). Simultaneously visualized fluorescence channel resulted from excitation by a 488 nm line of argon laser, representing the signals for SOSG fluorescence detected by 505–525 nm emission filter set. The proper intensity of lasers was set according to unstained samples at the beginning of each experiment54 (link). The integral distribution of signal intensity (ranging from 0 to 4095 levels of brightness) within the images was evaluated using software FV10-ASW 3.0 Viewer (Olympus).

For the immunofluorescence analysis, the cells were plated onto coverslips coated with 0.1% gelatin (Sigma Aldrich) in 12-well plates and fixed with 3% paraformaldehyde (PFA) (Songon, Shanghai) at room temperature. The fixed cells were permeabilized with cold ethanol, phosphate-buffered saline (PBS) containing 0.2% Triton X-100, or PBS containing 0.5% saponin. Nonspecific binding sites were blocked by incubating the coverslips with 3% BSA for 20 min. The fixed cells were incubated with primary antibodies for 1–2 h and with the appropriate Alexa Fluor-conjugated secondary antibodies for 1 h before mounting. The slides were visualized under a confocal or fluorescence microscope. The images were further analyzed with the FV10-ASW 3.0 viewer software.
For live cell imaging, the cells were grown in 35-mm glass-bottom dishes (MatTek or Nest) and staged into a heated incubation chamber with CO₂. The cells were imaged using an Olympus FV-10 confocal microscope or a fluorescent microscope, and the images were acquired with the FV10-ASW 3.0 viewer (Olympus) or Metamorph (Molecular Devices) software. The image stacks or movie files were further processed with ImageJ software. A phase microscope was used for vacuole imaging and counting. The vacuole sizes were calculated with blind method in digitally enlarged images on a computer screen using a scale ruler.

Fluorescent images were obtained using a confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan). Quantification of cells or parameters of interest was conducted in a 1250 × 1250 µm field of view for vessel density and %LV area, and a 200 × 200 µm field of view for counting EdU⁺ cells using FV10-ASW 3.0 Viewer (Olympus). To quantify the fluorescent intensity of indicated antibodies (Tfe3, CD31, Pdpn, Vegfr3, and Lyve1) using the Image J software (NIH, Bethesda, MD, USA), we measured the “Mean” values in randomly chosen four 50 × 50 µm fields of view in each image and were averaged. Blood-filled lymphatics were counted as the number of cross sections of LV tubes containing one or more erythrocyte in the entire embryo or tail section in scanned images. To get the serial images for z-stack slices, multiple slices horizontally imaged from the same field of view at 0.3-μm intervals were captured.

Fluorescent images were obtained using a confocal laser scanning microscope (Olympus, FV3000). Obtained images were analysed using FV10-ASW 3.0 Viewer (Olympus). Cells or substances of interest were quantified in the entire area of the epiphysis, in a 600 × 600 µm² field of view (FOV; to count OSX⁺ and c-Kit⁺ cells and to quantify relative intensity of immunofluorescence), or in a 1-mm length of the vascular front (to count tip-cell filopodia). To measure the distance between HSCs and ECs, two HSCs were randomly picked per scanned image. ImageJ software (the US National Institutes of Health) was used to quantify the indicated areas in scanned images.