After 24 h of culture, adhered cells on the films were fixed with a 4% paraformaldehyde solution (Sigma-Aldrich, Darmstadt, DE) for 20 min, permeabilized with 2% BSA solution with 0.1% Triton X100 (Sigma-Aldrich, Darmstadt, DE) for 1 h, and stained with fluorescein-conjugated phalloidin (Sigma-Aldrich, Darmstadt, DE) for 20 min and 4, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Darmstadt, DE) for 5 min. The cells were visualized by fluorescence microscopy (IX-73 Olympus). The quantification of phalloidin-FITC, was made with Image J software and plots were obtained with GraphPad Prism version 6 for Windows.
Ix 73
Manufactured by Olympus
Sourced in Japan, United States
The IX-73 Olympus is a high-performance inverted microscope designed for advanced research applications. It features a sturdy and versatile design, providing a stable platform for various imaging techniques. The IX-73 supports a range of objectives and accessories, allowing for a diverse array of applications in fields such as cell biology, materials science, and more.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde solution, by Merck Group (1 mentions)
Fluorescein conjugated phalloidin, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fastcam sa5, by Photron (1 mentions)
Anti smad2 3 5678s, by Cell Signaling Technology (1 mentions)
Anti col1a1 72026, by Cell Signaling Technology (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti il 1β, by Proteintech (1 mentions)
Dylight 488, by EarthOx (1 mentions)
Anti asc, by Cell Signaling Technology (1 mentions)
Anti il 18, by Proteintech (1 mentions)
Anti caspase 1, by Proteintech (1 mentions)
Rabbit anti nlrp3, by Proteintech (1 mentions)
Dapi solution, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
6 protocols using ix 73
1
Fluorescent Staining of Cultured Cells
2
Cell Culture on Rough PDMS Surfaces
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To culture cells on the rough PDMS surfaces, a 5 cm diameter Petri dish and 2 × 2 cm2 rough PDMS surface is thoroughly sterilized using 70% ethanol spray and then under the UV light for 30 min; the surface is placed in the Petri dish (Fig. 1d ). Each of the two cell lines, at a concentration of ∼2 × 105 mL−1 is seeded, and the Petri dish is covered with an ethanol-cleaned glass slide leaving a small air gap. Then the setup is gently placed inside the CO2 incubator. To monitor cell attachment, growth, and proliferation, the images of the cells on the surface is captured after 24, 48, and 72 h under an inverted microscope (IX 73 Olympus, Japan) with 5X-60× objective lenses and a high-resolution camera (FASTCAM SA5, Photron, UK) (Fig. 1d ). To monitor the initial progress in cell attachment, the images of the cells are also captured for the first 30 min. The length of the cells at different surface energy and roughness are measured to characterize the cell attachment and growth. The number of cells is counted at different time points using Image J software to quantify cellular proliferation.
3
Immunofluorescence Characterization of HSC-ADSC Coculture
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HSCs cocultured with ADSC were fixed with 4% paraformaldehyde solution for 1 h, and permeabilization was performed with 2% bovine serum albumin (BSA, Sigma-Aldrich) solution with 0.1% Triton X-100 for 20 min. This was followed by overnight incubation at 4 °C with specific antibodies: anti-α-SMA (sc-53015, Santa Cruz Biotechnology, Dallas, TX, USA), anti-SMAD2/3 (5678S, Cell Signaling), anti-COL1A1 (72026, Cell Signaling), and antifibronectin (sc-69681, Santa Cruz Biotechnology). Samples were incubated with specific anti-rabbit/anti-mouse secondary antibodies (Thermo Fischer Scientific) conjugated with AF 546 and AF 488, respectively, and further were stained with Hoechst 33342 (Sigma-Aldrich) for 5 min to visualize the nuclei in blue. Samples were visualized by fluorescence microscopy (IX-73 Olympus).
4
Immunofluorescence Analysis of Inflammasome Proteins
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With regard to the immunofluorescence method, myocardial tissue slices were incubated with the following primary antibodies: Rabbit anti-NLRP3 (1:100, Proteintech, 19771-1-AP, United States), anti-Caspase 1 (1:50, Proteintech, 22915-1-AP, United States), anti-IL-1β (1:200, Proteintech, 26048-1-AP, United States), anti-IL-18 (1:400, Proteintech, 10663-1-AP, China), anti-ASC (1:800, Cell Signaling, #67824, United States).
The slices were then washed and detected with appropriate Fluor dye, DyLight 488 (1:50, EarthOx, E032220-01, United States), as secondary antibodies followed by counterstaining with DAPI in the dark. Afterward, immunofluorescent signaling was observed with a fluorescence microscope (IX73 Olympus). Digital images and data were recorded and analyzed by applying ImageJ (NIH).
The slices were then washed and detected with appropriate Fluor dye, DyLight 488 (1:50, EarthOx, E032220-01, United States), as secondary antibodies followed by counterstaining with DAPI in the dark. Afterward, immunofluorescent signaling was observed with a fluorescence microscope (IX73 Olympus). Digital images and data were recorded and analyzed by applying ImageJ (NIH).
5
Immunofluorescence Quantification of Lineage Markers
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Samples were fixed with a 4% paraformaldehyde solution for 20 min, permeabilized with 2% BSA solution with 0.1% Triton X100 for 1 h. The samples were then incubated with osteopontin antibody (for osteogenic differentiation) or perilipin antibody (for adipogenic differentiation) for 12 h at 40 °C, after which they were incubated with anti-mouse FITC conjugated secondary antibody (SantaCruz, Heidelberg, Germany) for 1 h. Cell nuclei were stained with DAPI solution (Thermo Fisher Scientific, Waltham, MA, USA) and the cells were visualized by fluorescence microscopy (IX-73 Olympus). The quantification of perilipin and OPN levels was made with Image J software and plots were obtained with GraphPad Prism version 6 for Windows.
6
Hairy Root Microscopy Protocol
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Spent medium from the alpha-L-iduronidase (IDUA) expressing hairy root line after induction of secretion were analyzed as previously described (Cardon et al., 2019) .
Microscopy and photographs of B. rapa rapa hairy root lines B. rapa rapa hairy root fragments were excised from the liquid culture, mounted on microscopy slides in water or in toluidine blue, and analyzed with an inverted microscope (IX73 OLYMPUS). Macroscopic pictures were captured using a Nikon D5200 camera.
Microscopy and photographs of B. rapa rapa hairy root lines B. rapa rapa hairy root fragments were excised from the liquid culture, mounted on microscopy slides in water or in toluidine blue, and analyzed with an inverted microscope (IX73 OLYMPUS). Macroscopic pictures were captured using a Nikon D5200 camera.
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