We cultivated SW1271 cells on the slides, washed them with PBS, and fixed them in 4% paraformaldehyde. After permeabilization, the hybridization was performed overnight with the lncRNA BANCR fluorescence in situ hybridization (FISH) probes (5'-ACAGGACTCCATGGCAAACG-3'). The cytoskeleton was stained with a fluorescent staining reagent for vinculin and cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at room temperature. Specimens were analyzed on a Nikon inverted fluorescence microscope (Nikon, Japan).
Inverted fluorescence microscope
Manufactured by Nikon
Sourced in Japan, United States, China, Italy
The Inverted Fluorescence Microscope is a laboratory instrument designed to observe and analyze fluorescent samples. It features a reversed optical design, where the light source and objectives are positioned below the stage, allowing for the examination of cells and tissues cultured in petri dishes or microplates. The microscope utilizes fluorescence excitation and emission to enable the visualization of specific cellular structures or molecules labeled with fluorescent dyes.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (17 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (10 mentions)
Matrigel, by BD (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Fura 2 am, by Thermo Fisher Scientific (5 mentions)
Flow cytometry, by BD (5 mentions)
Image pro plus 6, by Media Cybernetics (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Digital camera, by Nikon (4 mentions)
Vimentin, by Abcam (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
4 6 diamino 2 phenylindole dapi, by Merck Group (3 mentions)
Hoechst 33258, by Beyotime (3 mentions)
In situ cell death detection kit, by Roche (3 mentions)
Antifading prolong gold solution, by Thermo Fisher Scientific (3 mentions)
Basic research software, by Nikon (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (3 mentions)
Paraformaldehyde (pfa), by Solarbio (3 mentions)
437 protocols using inverted fluorescence microscope
1
Localization of lncRNA BANCR in SW1271 Cells
2
Immunohistochemical Analysis of Retinal Proteins
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Immunohistochemistry was performed according to previous publications [53 (link), 54 (link)]. Retinal sections were mounted onto coated glass slides. Sections were rehydrated in PBS for 20 min then immersed in a blocking buffer containing 2 % BSA, 0.5 % Tween-20 and 0.05 % Triton-X 100 for 1 h. Primary antibody for PK2 (Hamster monoclonal, 1:200, Roche Inc.) or OPN4 (Affinity purified rabbit polyclonal, 1:200, Millipore Inc.) was added to the sections overnight at 4 °C. Slides were washed with PBS containing 0.5 % Tween-20 five times for 5 min each. Anti-rabbit or anti-hamster secondary antibodies (Alexa Fluor 488 or 555 1:2000; Invitrogen Inc.) were then applied, followed by incubation with 10 μg/ml Hoechst 33342 (Invitrogen Inc) for 5 min at room temperature to stain the nucleus. Sections were viewed under a Nikon inverted fluorescence microscope (Model TE-2000U; Nikon Inc, Tokyo, Japan). Images were captured with a SPOT digital camera (Diagnostic Instruments, Inc, Sterling Heights, MI). Immunofluorescence intensity was quantified with Image J. For DAB (3,3′-diaminobenzidine) immunostaining, sections were incubated with anti-PK2 antibody (Hamster monoclonal, 1:500 dilution) antibody, followed by incubation with biotinylated anti-hamster secondary antibody. Color development of DAB immunostaining was carried out with the standard ABC method [52 (link)].
3
Measurement of Intracellular Oxidative Stress
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MEFs were cultured in 0.1% gelatin-coated 3-cm Petri dishes. After treatment with 0.1% DMSO for the indicated time, cells were washed with warm Hanks’ balanced salt solution (HBSS: Gibco-Thermo Fisher Scientific) and then loaded with 25-μM chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H2DCFDA; C-6827, Life Technologies) in complete growth medium for 10 min at 37°C in the dark. After loading, cells were washed twice with HBSS and examined using a Nikon inverted fluorescence microscope. Three to five fields were selected for imaging with a 10× objective lens and quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA). The value of WT basal (set as 1.0) was used to normalize the results.
4
Measuring Foam Bubble Diameter
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The bubble diameter of foam was determined, as described previously [22 (link)], by using a Nikon inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan) and calculated by using software (NIS-Elements BR4.00.12). The bubble diameter was measured twice per day for 4 consecutive days. Data were collected from at least 50 bubbles each time.
5
Immunofluorescence Labeling of Cell Adhesion Components
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For immunofluorescence labeling of cell adhesion components, SF295 cells were seeded on ZnO thin film substrates at the indicated densities. Cells were fixed with 4% (w/v) paraformaldehyde for 10 minutes, washed three times with PBS, and then permeabilized and blocked by incubating with PBS containing 1% horse serum and 0.1% Triton X-100 for 30 minutes. FA complexes were analyzed by incubating overnight at 4°C with anti-vinculin (1:400; Sigma) or phosphospecific anti-FAK [pY397] (1:1000; Invitrogen) antibodies. The next day, the cells were washed three times with blocking solution and then incubated with the appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:2000, Jackson Immunoresearch) containing 4',6-diamidino-2-phenylindole (DAPI, 1:1000; Sigma) for 3 hours. After washing with blocking solution (PBS/1% horse serum), the samples were mounted on glass slides for subsequent imaging processing. All fluorescent images were acquired on a Nikon inverted fluorescence microscope (Nikon Instruments, Japan).
6
Fluorescence Microscopy Imaging Setup
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A Nikon inverted fluorescence microscope was utilized throughout these experiments (Nikon, 2021 ). A Lumencor Sola Light Engine (Lumencor, 2021a ) solid-state illumination provides fluorescent light combined with excitation- and emission filters in the microscope at 575 and 641 nm, respectively. Bright-field images are provided by the build-in lamp in the microscope. Images are captured with an Andor Neo sCMOS model DC-152Q-COO-FI camera (Lumencor, 2021b ).
7
Liver Lipid Visualization and Quantification
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H&E and oil red O staining were performed in liver tissue. Liver fat accumulation was observed under a microscope (Olympus, Tokyo, Japan). The cells were incubated with TET1 antibody (11000) (GeneTex, CA, USA) for 2 h at room temperature and then incubated with secondary antibody and fat fluorescent dye BODIPY493/503 (Thermo Fisher Scientific, MA, USA) for 1 h, and the nuclei were stained with DAPI (Beyotime shanghai, China) for 1 min. Results were observed under a Nikon inverted fluorescence microscope (Nikon, Tokyo, Japan).
8
Fluorescence Microscopy Imaging Protocols
9
Quantifying Cell Proliferation with CCK-8 and EdU
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Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) according to the manufacturer’s instructions (Dojindo, Kumamoto, Japan). U87MG or U251 (at 2 × 103 cells/well) were incubated in 96-well plates for 24, 48, and 72 h. CCK-8 solution (10 μL) was added to each well, the plates were incubated for 1 h at 37 °C, and absorbance at 450 nm wavelength (OD450) was measured in a Microplate Reader (Bio-Rad). For the EdU assay, cells were incubated with 200 μL of 5-ethynyl-20-deoxyuridine (Ribo-Bio; Guangzhou, China) for 2 h at 37 °C. Cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.4% Triton X-100 for 10 min, and incubated with Apollo® reagent (100 μL) for 30 min. Nuclei were stained with DAPI, and representative images obtained with a Nikon inverted fluorescence microscope.
10
Proliferation Assay of SF295 Cells on ZnO Films
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The proliferation rate of SF295 cells on ZnO films was determined using a Click-iT EdU Alexa Fluor 488 Imaging Kit (Molecular Probes, CA). SF295 cells were seeded on glass (control) or ZnO thin films. The next day, the cells were pulsed-labeled for 1 hour with EdU (10 μM). After replacing the cell growth medium with fresh medium, the label was chased by incubating cells for an additional 7–11 hours. After the chase period, the cells were fixed with 4% paraformaldehyde for 15 minutes and then stained with Click-iT reaction reagents as described by the manufacturer (Invitrogen, CA). In brief, fixed cells were rinsed twice with PBS/3% bovine serum albumin (BSA) and permeabilized with PBS/0.5% Triton X-100 for 20 minutes. The permeabilization buffer was replaced with 500 μL of Click-iT reaction cocktail to detect proliferating cells. The nuclei were stained with Hoechst 33342 following standard protocols. All staining procedures were performed at room temperature, and all fluorescence images were obtained using a Nikon C1 laser-scanning confocal microscope system equipped with a Nikon inverted fluorescence microscope (Nikon, Japan).
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