Total cell number counting, BrdU incorporation, cell cycle analysis, TUNEL, Annexin V, foci formation, soft agar colony formation assay, 3D-matrigel culture, in vitro cell motility (migration, invasion and wound healing) assays and tumorsphere formation assay were carried out as previously described [55 (link)–57 (link)]. All the images were taken using a phase microscope (Olympus, Tokyo, Japan). The CD133 positive population in HCC cells was examined by using PE-conjugated monoclonal mouse anti-human CD133/1 (AC133, Miltenyi Biotec; Auburn CA).
Phase microscope
Manufactured by Olympus
Sourced in Japan
The Phase microscope is an optical microscope that uses phase-contrast illumination to enhance the visibility of transparent or unstained specimens. It works by converting small differences in the refractive index within a specimen into corresponding differences in brightness, allowing for improved contrast and detailed observation of cellular structures and other biological samples.
Automatically generated - may contain errors
7 protocols using phase microscope
1
Comprehensive Analysis of Cancer Cell Properties
2
Immunohistochemical Staining of Tumor Markers
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The immunohistochemical experiment was performed according to our previous study (Bai et al., 2021 (link)). All procedures were performed according to the guidelines of the National Institutes of Health regarding the use of human tissues. First, the 4 µm sections were baked at 63 °C for 2 h, dewaxed in xylene and rehydrated in gradient alcohol. After performing antigen retrieval, sections were treated with 3% hydrogen peroxide (H2O2) for 10 min and incubated with normal goat serum (ZSGB Biotech, Beijing, China) for 1 h at room temperature. Next, tissue sections were incubated overnight at 4 °C with the following primary antibodies: anti-CCR9 (1:100), anti-E-cadherin (1:200), anti-vimentin (1:200), anti-Ki67 (1:1,000), and anti-SLUG (1:50). Then, antibody binding was detected by horseradish peroxidase streptavidin (#SP-9000; ZSGB Biotech, Beijing, China). Finally, the sections were developed using diaminobenzidine (DAB, #ZLI-9018; ZSGB Biotech, Beijing, China) and counterstained with hematoxylin. The images of staining sections were photographed using a phase microscope (Olympus Corporation, Tokyo, Japan), and the integral optical density (IOD) was evaluated by Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA).
3
Isolation and Measurement of Salivary Glands
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Flies were put into a plastic cup and placed in a −80°C freezer for 4 min, then individually put on a dissecting dish using Ento-pins to secure them under a Bausch & Lomb dissecting microscope. Approximately, 300 µl of PBS was used to flood the abdomen of the fly, and stainless steel no. 5 forceps used to dissect the abdomen and remove the salivary glands. To best remove the glands, the salivary glands were grasped in the neck region by the common duct of the salivary glands. Each pair of glands was put into 1.5-ml microcentrifuge tubes with 50 µl of PBS. Glands from infected flies used for measurements (N = 46 for both sugar-fed and N = 44 for protein-fed flies) were put on a glass slide in PBS and measured on an Olympus Phase microscope with an ocular micrometer. Two measurements were taken: one consisted of measuring the terminal bulb of the salivary gland (Fig. 1 , DM), while the other was to measure the narrower width, which was just prior to the bulb (Fig. 1 , asterisk). Images were taken with an AxioCam ERc5s with Zen imaging program.
4
Scratch Assay for Cell Migration
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The transfected OSCC cells were seeded onto a six-well plate at a density of 5 × 105 cells. Once the cells adhered stably to the plate, they were treated with the corresponding inhibitors based on their respective groups. A 200-μL pipette tip was used to create a vertical scratch across the bottom of the plate. The scratch was then rinsed three times with PBS to remove any remaining cells, after which the cells were cultured in a serum-free medium. Images were captured at 0 h and 12 h using a phase microscope from Olympus Corporation, Tokyo, Japan. Finally, Image J software was utilized for image analysis.
5
Scratch Wound Assay for Cell Migration
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SACC-LM cells (1 × 106) were seeded into six-well plates. When the density of cells grew to 70–80%, 200 μL CCL25 at concentrations of 0, 100, and 200 ng/ml was added to the wells. A 200-μL pipette tip was used to make a straight artificial wound scratch, and the cells were incubated for 24 h at 37 °C. Then, the SACC-LM cells that migrated across this straight scratch were observed by a phase microscope (Olympus Corporation, Tokyo, Japan) and evaluated by ImageJ 1.42 software (National Institutes of Health, Rockville, MD, USA).
6
CCL25 Impacts SACC-83 Cell Colony Formation
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1 × 102 SACC-83 or SACC-LM cells were seeded in 60 mm dishes and cultured in DMEM/F-12 with 10% FBS and 1% Penicilin-Streptomycin Solution in a humidified incubator at 37°. After adhesion, the cells were treated with 0, 50, 100 and 200 ng/ml CCL25 respectively. Fresh DMEM/F-12 media with 10% FBS were replenished every 5 to 6 days. After 10 or 14 days, the colonies were fixed with 4% PFA at room temperature for 20 min, and then stained with Giemsa stain (Solarbio, Beijing, China) for 15 min. Finally, the dishes were washed with PBS for 3 times and photographed by a phase microscope (Olympus Corporation, Tokyo, Japan).
7
Matrigel Tube Formation Assay
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According to our previous study [16 (link)], the cool 96-well plate was coated with 50 ul of Matrigel (BD Biosciences) and placed in a 37 °C incubator for 45 min. Next, HUVECs were seeded in triplicate for 10 h and capillary-like structures were quantified under a phase microscope (Olympus).
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