Dm il inverted microscope

Slide images were acquired using bright field microscopy using a Leica DM4000B upright microscope (Leica Microsystems, Wetzlar, Germany) fitted with either a HC PL apochromatic 10X objective with 0.40 numerical aperture (NA), HC PLAN apochromatic 20X objective with 0.70 NA, or HCX PL apochromatic 40X objective with 0.85 NA. Image capture was performed using an Axiocam 105 Color 5-megapixel CMOS digital microscope camera (Carl Zeiss, Jena, Germany) with a 0.55X magnification c-mount attachment. A PC with Windows 10 Enterprise operating system was used to run Zen 2 Core software (Carl Zeiss) for image capture. Slides stained for VEGFR-2 were visually compared with positive control clinical KS references and no-primary controls to qualitatively verify expression of the receptor. Cell culture images were acquired using bright field microscopy with phase contrast, using a Leica DMIL inverted microscope (Leica Microsystems, Wetzlar, Germany) fitted with a HI PLAN achromatic 10X objective with 0.25 NA. Stand-alone image capture was performed using an Axiocam ERc 5s Color 5-megapixel CMOS digital microscope camera (Carl Zeiss, Jena, Germany) with a 0.55X magnification c-mount attachment.

MDCK cells were grown to 70 to 80% confluence on coverslips. Cells were propagated with influenza A/WSN/33 wild-type virus or mutant viruses at an MOI of 10 in fresh MEM containing 0.2% FBS and 1 μg/ml TPCK-treated trypsin. A total of 20 μM FA-6005 or DMSO was added at the indicated time points. Infections were stopped at the indicated time points by fixation with 500 μl of 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 15 min. After fixation, the cells were washed with PBS three times and permeabilized with 1 ml 70% ethanol at 4°C overnight. Custom Stellaris fluorescence in situ hybridization (FISH) probes for the detection of vRNA and mRNA of segment 5 of influenza virus were designed by utilizing the Stellaris FISH probe designer (Biosearch Technologies Inc., Petaluma, CA). The assay for simultaneous immunofluorescence (IF) microscopy and FISH was performed according to the manufacturer’s instructions (available at www.biosearchtech.com/stellarisprotocols). The slides were stored at 4°C overnight before being sealed with nail polish to prevent drying. The slides were observed and photographed using a Leica DMIL inverted microscope equipped with a DC300F digital imaging system (Leica Microsystems, Germany) or an LSM710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

For the enrichment of STRO-1+ cells (+), dental-pulp cells were incubated with a mouse (ms) anti-human STRO-1 antibody (2 µg/10⁶ cells; R&D Systems, Minneapolis, MN, USA) diluted in cold rinsing buffer (phosphate-buffered saline (PBS), Life Technologies) with 2 mM EDTA (Sigma Aldrich) containing 0.5% bovine serum albumin (BSA; Sigma Aldrich) for 30 min at 4 °C. After washing, cells were incubated with anti-ms IgM microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4 °C. After a final washing step, (+) cells were brought into a magnetic field (MACs Multistand Miltenyi Biotec) using the MiniMACS™ separator (Miltenyi Biotec) containing paramagnetic beads. Subsequently, cells were removed from the magnetic field and collected in 1 mL buffer, using a plunger and kept in culture medium at 37 °C with 5% CO₂.
For the retrieval of colony-derived cells (c), dental-pulp cells were plated in limiting dilution. Emerging colonies were photographed using a Leica DMIL inverted microscope (Leica Microsystems, Wetzlar, Germany) connected to a Leica D-Lux3 CCD camera (Leica Camera, Solms, Germany), and cells were subsequently collected.
For the enrichment of colony-derived STRO-1+ cells (c/+), dental-pulp cells were plated in limiting dilution, harvested from colonies, and subsequently enriched based on their STRO-1 expression, as described above.

To measure migration, we plated 1,000,000 stably transfected control vector or sh-HAMP SMMC-7721 cells and HepG-2 cells per well on six-well plates in a 2-mL volume for 24 h. We then generated a wound in the cell layer using a 200-µL tip, and cells in this area were removed via PBS rinsing. Low-serum media were then added to the cells (DMEM + 0.5% FBS), and a Leica DMIL inverted microscope was used to image cells at 0 and 24 h. The area of this wound was measured in Image J, and the degree of wound closure was calculated as follows: wound closure = wound area at 0 h − wound area at 24 h.