The transfected BCa cells (~2×105 cells/ml) were inoculated in the upper chamber (200 µl/well) of a Transwell device (Corning, Inc.). The lower chamber was incubated with culture medium containing 10% fetal bovine serum (60 µl/well) and incubated for 24 h at 37°C. The cells were washed with PBS, fixed by the addition of paraformaldehyde for 20 min at 20°C, and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 10 min at 20°C. The number of migrating cells was observed under a fluorescent microscope (Olympus Corporation) for 10 min. For the Transwell invasion assay, the pre-cooled culture medium was used to dilute the Matrigel matrix (0.8 µm; Corning, Inc.) gel, which was added to the upper chamber and incubated for 5 h at 37°C. The BCa cell suspension was then added, and the subsequent experimental steps were the same as those for the cell migration assay. The number of invading cells was determined under a fluorescent microscope (Olympus Corporation). Images were captured under a fluorescent microscope at ×200 magnification (Olympus Corporation) to calculate cell migration and invasion using ImageJ software (Version 1.45s; National Institutes of Health).
Fluorescent microscope
Manufactured by Olympus
Sourced in Japan, United States, Germany, China, Austria
The Olympus Fluorescent Microscope is an optical instrument that uses fluorescence to visualize and analyze biological samples. It allows for the observation of cellular structures and processes by illuminating the sample with a specific wavelength of light, causing fluorescent molecules within the sample to emit light at a different wavelength. This enables the identification and localization of specific molecules or structures within the sample.
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Lab products found in correlation
Image pro plus 6, by Media Cybernetics (18 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (16 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (15 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (15 mentions)
Triton x 100, by Merck Group (12 mentions)
Matrigel, by BD (10 mentions)
Hoechst 33342, by Olympus (9 mentions)
Edu assay kit, by RiboBio (8 mentions)
Click itr edu kit, by Olympus (7 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
In situ cell death detection kit, by Roche (7 mentions)
Dp70 camera, by Olympus (7 mentions)
Triton x 100, by Beyotime (6 mentions)
5 ethynyl 2 deoxyuridine (edu), by RiboBio (6 mentions)
Image pro plus software version 6, by Media Cybernetics (5 mentions)
Paraformaldehyde (pfa), by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (5 mentions)
Cell light edu dna cell proliferation kit, by RiboBio (5 mentions)
Cryostat, by Leica (5 mentions)
Hoechst 33342, by Beyotime (5 mentions)
859 protocols using fluorescent microscope
1
Transwell Migration and Invasion Assay for Bladder Cancer
2
Immunofluorescence Analysis of EPCs and NSCs
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For the in vitro experiments, the EPCs or NSCs were grown on glass slides in 6-well plates. The cells were fixed in 4% paraformaldehyde for 30 min at room temperature. Immunostaining was performed using mouse monoclonal anti-VEGR2 (1:150; Biorbyt, Ltd.), polyclonal rabbit anti-CD133 (1:150) and/or polyclonal rabbit anti-nestin (1:150; Sigma-Aldrich; Thermo Fisher Scientific, Inc.) and FITC-conjugated anti-rabbit or anti-mouse IgG (1:200; Sigma-Aldrich; Thermo Fisher Scientific, Inc.) and tetra methyl rhodamyne iso-thiocyanate anti-rabbit or mouse IgG (1:200; Sigma-Aldrich; Thermo Fisher Scientific, Inc.) secondary antibodies were utilized, and counterstaining for nuclei was performed using 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride. Immunofluorescence was visualized using a fluorescent microscope (Olympus Corporation, Tokyo, Japan). The results were based on three independent analyses.
To determine the uptake of Dil-Ac-LDL and binding of FITC-UEA-1, the EPCs were incubated with Dil-Ac-LDL overnight at 37°C, and fixed with 4% paraformaldehyde for 20 min. The cells were then incubated with FITC-UEA-1 for 1 h at 37°C, and examined under a fluorescent microscope (Olympus Corporation).
To determine the uptake of Dil-Ac-LDL and binding of FITC-UEA-1, the EPCs were incubated with Dil-Ac-LDL overnight at 37°C, and fixed with 4% paraformaldehyde for 20 min. The cells were then incubated with FITC-UEA-1 for 1 h at 37°C, and examined under a fluorescent microscope (Olympus Corporation).
3
Hoechst 33342 Staining for Apoptosis Evaluation
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Hoechst 33342 staining was carried out to observe morphological characteristics of apoptotic cells. Briefly, cells were seeded on coverslips in 24-well plates at a density of 5 × 104/ml per well and incubated overnight. Then, the cells were treated with 100 μM Gli for another 24 h. After removal of the medium, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100, and stained with Hoechst 33342 solution (5 μg/ml) for 10 min at room temperature in the dark. Subsequently, the slides were rinsed twice with PBS and photographed with a fluorescent microscope (Olympus Optical Co., Ltd., Tokyo, Japan) for morphological changes in the nucleus.
4
Cell Proliferation Viability Assay
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This assay was performed according to the method of a previous study [45 (link)]. To assess the proliferation viability of cells, the EdU assay was carried out with a BeyoClickTM EdU−555 detection kit (Beyotime, Shanghai, China). Transfected RASFs or MH7A cells were seeded in twelve-well plates and incubated with complete medium for 12 h. After incubation with 50 mM EdU for 6 h, the cells were fixed and stained for 30 min. The nucleic acid was stained with Hoechst 33342. All images were captured with a fluorescent microscope (Olympus Optical Co., Ltd., Tokyo, Japan).
5
Comet Assay for Nanoparticle-Induced DNA Damage
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Nanoparticle treated cells were embedded in 0.5% low melting agarose (Sigma Aldrich) layer between 1.0% normal melting agarose (Sigma Aldrich) and 0.5% normal melting agarose. The cells were lysed with high salt and detergent concentrations (100 mM EDTA (Sigma Aldrich), 2.5 M NaCl (Sigma Aldrich), 10 mM tris base (Bio Rad), 1% Triton X-100 (Sigma Aldrich), adjusted to pH 10) for 1 h. DNA was allowed to unwind (1 mM EDTA, 10% DMSO, 300 mM NaOH (Sigma Aldrich), pH 13) for 20 min and then subjected to electrophoresis in the same solution as for unwinding (25 V, 300 mA) for 15 min. After electrophoresis, the alkalis in the gels were neutralized by rinsing the slides in a neutralization buffer (0.1 M Tris pH 7.5) for 5 min. The slides were treated with methanol for 10 minutes, stained with 45 μl of 20 μg/ml ethidium bromide solution and viewed under a fluorescent microscope (Olympus Optical Co. Ltd). 1000 cells were analyzed and % tail DNA was measured to evaluate the extent of DNA damage. Results were expressed as means of at least three replicates ± standard error.
6
Chlorogenic Acid Bax and Bcl-2 Immunofluorescence
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About 2 × 10 5 of cells were poured into each one of the 24 well tissue culture plates and incubated for 24 h. After adding chlorogenic acid solution (200 μM), incubation was carried out for 48 h. 4% paraformaldehyde was added to fix the cells, and the contents were washed with PBS after 20 min and for two times. 2 N HCl was added and then removed after 20 min. Borate buffer was added and then washed with PBS. Afterward, 3% of Triton-X 100 was added to the plates and washed with PBS after 20 min. Next step was adding 10% normal goat serum and then removing it after 20 min. Then, the cells were incubated with polyclonal rabbit antibodies Bax (1:100, Santa Cruz Biotechnology, SC493) and polyclonal rabbit antibodies Bcl-2 (1:100, Santa Cruz Biotechnology, SC783) all over the night at 4°C. After the overnight incubation, coverslips were washed with PBS twice. The cells were incubated for 2 h with anti-mouse IgG (1:200, Abcam, Ab97022) and anti-rabbit IgG (1:200, Biorbyt, ORB180726) for 90 min in the darkness. Ultimately, the cells were washed with PBS, followed by the addition of PI for 20 min. The cells were visualized using a fluorescent microscope (Olympus Optical), and images were acquired with a magnification of ×400. Finally, the images were analyzed for markers' verification.
7
Immunofluorescence Staining of IL-35 and MDSCs
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We performed immunofluorescence staining for IL-35 and M-MDSCs in the skin of patients with psoriasis and healthy controls and for MDSCs and iNOS+ MDSCs in murine skin lesions. Mice were treated with IL-35; the back skin was obtained the day after the last treatment, frozen, and then stained. Samples were incubated with primary antibodies (rat anti-human p35 and CD14, anti-mouse CD11b and iNOS, rabbit anti-human EBI3+ and HLA-DR, anti-mouse Gr-1) (Abcam) at 4°C overnight, followed by directly-labelled IgG anti-rat or rabbit antibodies (ZsBio, Beijing, China) for 1 hour. Tissue sections were examined under a fluorescent microscope (Olympus Optical, Tokyo, Japan). Images were captured at zoom magnification.
8
Hippocampus Immunohistochemistry of Rats
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After training and testing, rats were sacrificed and their brains were removed, fixed in 4% paraformaldehyde, and dehydrated in sucrose solution (20%–30%). The tissue was embedded in optimal cutting temperature compound and cut into 10-μm sections. The sections underwent antigen retrieval with citric acid retrieval solution and heating for 10 minutes before being blocked with 1% bovine serum albumin (Sangon Biotech, Shanghai, China). Thereafter, the sections were incubated with primary antibodies against postsynaptic density protein (PSD95; 1:100; Affinity, Changzhou, China; AF7839), microtubule-associated protein 2 (MAP2; 1:200; Affinity; AF4081), and F-actin (1:25; Abcam, Cambridge, UK; ab205) at 4°C overnight, followed by incubation with Cy3- or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA or Abcam) for 60 minutes at room temperature. After being counterstained with 4′,6-diamidino2-phenylindole (DAPI; Aladdin, Shanghai, China), the sections were observed and images of the CA1 region of the hippocampus were captured using a fluorescent microscope (Olympus Optical Co., Tokyo, Japan).
9
Comet Assay for DNA Damage Evaluation
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Genomic DNA damage was measured by using comet assay. Briefly, BV2 cells after IH exposure were embedded in 0.6% (w/v) low melting point agarose and then lysed in 2.5 mol NaCl, 10 mmol Tris-HCl (pH 10), 100 mmol EDTA, 1% (v/v) Triton X-100 at 4 °C overnight. Denaturation (20 min) followed by electrophoresis (20 min at 25 V and 300 mA) was performed in a solution of 0.3 mol NaOH, 1 mmol EDTA. Then the slides were washed, stained with SYBR Green (Thermo Fisher Scientific, Inc. Lafayette, CO, USA) and observed using a fluorescent microscope (Olympus Optical Co., Ltd. Tokyo, JP). Slides were analysed using the Komet 5 analysis software (Kinetic Imaging, Liverpool, UK).
10
Apoptosis Quantification via TUNEL Assay
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One-step TUNEL Apoptosis Detection Kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) was used to assess the cell apoptosis. After the cells were fixed and permeabilized according to the manufacturer's instructions, each sample was added dropwise with 50 μl TUNEL reagent and incubated at 37 C for 60 min in the dark. Nuclei were stained with DAPI. The cells were sealed with sealing solution containing an anti-fluorescence quencher, observed using a fluorescent microscope (Olympus Optical Co., Ltd., Tokyo, Japan), and the results recorded. The apoptotic index was defined as the ratio of the number of TUNEL positives per field of view (40-50 cells)/DAPI Â 100%.
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