The intracellular ROS production was measured by using 2′,7′-dichloro-fluorescin diacetate (DCFH-DA) (Abmole, Shanghai, China). Caprine EECs were seeded in 12-well cell culture plates (Shanghai Sangon Biotech, Shanghai, China) and infected with N. caninum tachyzoites with a MOI of 3:1 (parasite:cell) for 48 h. The cells were washed with PBS and incubated with DCFH-DA (10 μM) in the dark for 20 min at 37 ℃. After washing the cells with serum-free DMEM/F12 medium for 5 min, the fluorescence intensity was detected under an inverted fluorescence microscopy (Leica Microsystems, Wetzlar, Germany). The fluorescence intensity of green (excitation/emission wavelengths of 488/530 nm) represented the intracellular ROS levels.
Inverted fluorescence microscopy
Manufactured by Leica
Sourced in Germany
The Inverted fluorescence microscopy is a type of microscope that illuminates and observes specimens from below. It allows for the visualization of fluorescently labeled samples, enabling the study of cellular structures and processes.
Automatically generated - may contain errors
Lab products found in correlation
12 well cell culture plates, by Sangon (2 mentions)
Matrigel, by BD (2 mentions)
Laser confocal microscopy, by Leica (2 mentions)
Dcfh da, by AbMole (1 mentions)
Jc 1 washing buffer, by Beyotime (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Hoechst 33342 staining, by Solarbio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Hoechst 33342 staining solution, by Beyotime (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Hoechst 33342, by Solarbio (1 mentions)
Chamber slide, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Dil ac ldl, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated lectin, by Merck Group (1 mentions)
Transwell chamber, by Corning (1 mentions)
24 protocols using inverted fluorescence microscopy
1
Measuring Intracellular ROS in Caprine EECs
2
Transwell Assay for Migration and Invasion
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Transwell assays were used for migration and invasion assays. For invasion assays, the lower chambers were precoated with 100 μL of Matrigel (BD Bioscience, San Jose, CA, USA) for 30 min before the addition of medium to the chambers. The transfected GC cells (1 × 106 cells/mL) were resuspended in RPMI 1640 medium. The upper chamber contained 100 μL of cell suspension medium, and 600 μL of complete medium was added to the bottom chamber. After incubating at 37 °C with 5% CO2 for 24 h, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. The cells that passed through the filter were photographed and counted by inverted fluorescence microscopy (Leica Microsystems GmbH, Wetzlar, Germany) in five randomly selected fields.
3
Glioma Cell Invasion Assay
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For invasion assays, the lower chambers were precoated with 100 μL of Matrigel (BD Bioscience, San Jose, CA, USA) for 30 min before the addition of medium to the chambers. The glioma cells (2 × 105 cells/mL) were resuspended in DMEM medium. The upper chamber contained 100 μL of cell suspension medium, and 600 μL of complete medium was added to the bottom chamber. After incubating at 37 °C with 5% CO2 for 24 h, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. The cells that passed through the filter were photographed and counted by inverted fluorescence microscopy (Leica Microsystems GmbH, Wetzlar, Germany) in four randomly selected fields.
4
Mitochondrial Membrane Potential Assay
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The MMP of intracellular mitochondria was monitored by using the mitochondrial membrane potential assay kit with JC-1 (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. Briefly, caprine EECs were seeded in 12-well cell culture plates (Shanghai Sangon Biotech, Shanghai, China) and infected with N. caninum tachyzoites with a MOI of 3:1 (parasite:cell) for 48 h. Then, cells were washed with PBS and incubated with JC-1 (1 ×) in the dark for 30 min at 37 ℃. After washing with the JC-1 washing buffer (Beyotime Biotechnology, Shanghai, China), cells were observed under an inverted fluorescence microscopy (Leica Microsystems, Wetzlar, Germany) to detect fluorescence of green (excitation/emission wavelengths = 490/530 nm) and red (excitation/emission wavelengths = 525/590 nm). The relative MMP was expressed as the ratio of red/green fluorescence intensities.
5
Hoechst 33342 Staining for Apoptosis
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To distinguish apoptotic from normal cells, Hoechst 33342 staining (Solarbio, China) was used for the qualitative analyses of PC12 cells [43 (link)]. Briefly, after treatment, cells are incubated with Hoechst 33342 for 15 min at room temperature, rinsed three times with phosphate-buffered saline (PBS), and then captured under an inverted fluorescence microscopy (Leica Microsystems, Wetzlar, Germany).
6
Hoechst and DAPI Nuclear Staining of PC12 Cells
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PC12 cells were cultured in 6-well plates before CRSE treatment. After treatments, the cells were washed twice with PBS, and 1 mL of Hoechst 33342 staining solution (Cat# C1025, Beyotime, Shanghai, China) was added to each well for a further incubation of 25 minutes at 37°C. The cells were then washed twice with PBS before incubation with 50 μg/mL DAPI (Cat# C1005, Beyotime, Shanghai, China) for 15 minutes at room temperature. Cells were then subjected to further visualization and analysis under an inverted fluorescence microscopy (Leica Microsystems, Wetzlar, Germany) and photographed.
7
Immunofluorescence Staining of Epithelial-Mesenchymal Transition
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After the indicated transfection, the cells were fixed with 95% ethanol for 20 min and permeabilized for 10 min on ice with 0.2% Triton X-100 (Sigma-Aldrich, China). Next, the cells were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. The cells were then treated with the corresponding primary antibodies at 4 °C overnight, followed by fluorochrome-conjugated secondary antibodies. DAPI (Sigma, China) was utilized for DNA staining, and the cells were then sealed after 30 min. Finally, image capture was performed using confocal laser microscopy (Leica, Germany) or inverted fluorescence microscopy (Leica, Germany). All antibodies used in this study are as follows. Primary antibodies: E-Cadherin (Cell Signaling Technology, Cat# 14472, 1:200), Vimentin (Cell Signaling Technology, Cat# 13116, 1:200), FBXO32 (abcam, Cat# ab168372, 1:200). Secondary antibodies for IF: anti-Rabbit IgG FITC-conjugated secondary antibody (Proteintech, Cat# SA00003-2, 1:200), anti-Rabbit IgG CY3-conjugated secondary antibody (Proteintech, Cat# SA00009-2, 1:100).
8
Immunohistochemical Analysis of CXCL2 in Lung Tissue
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After paraffin removal, lung tissue sections were washed by PBST and incubated with 3% H2O2 for 10 min. The sections were then blocked with 5% BSA at 37 ℃ for 1 h. After then, samples were incubated overnight with CXCL2 antibody（1:200 diluted in 5% BSA). Sections were washed with PBST and incubated with the corresponding secondary antibody at 37 ℃ for 1 h. The immunoreactivity of CXCL2 protein was visualized by a DAB substrate kit (#AR1022) and stained with hematoxylin. Finally, the fixed stained slides were photographed by inverted fluorescence microscopy (Leica, Germany). and the expression of CXCL2 was quantified by Image J software.
9
Multiplexed Immunofluorescence Staining Protocol
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For IF analysis, samples were incubated overnight with primary antibodies against Histone H3 (1:200), Myeloperoxidase (1:200), CD62P (1:200), CXCR2 (1:200), and Ly-6G (1:200). Subsequently, samples were washed with cold PBST and incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody (1:100) or Alexa Fluor 647-conjugated goat anti-rat antibody (1:100) or Alexa Fluor 488-conjugated donkey anti-Mouse secondary antibody (1:100) for 60 min. After staining the nuclei with Hoechst 33342 (C0031, Solarbio, China), images were acquired with multiple random fields by inverted fluorescence microscopy (Leica, Germany). and target proteins were quantified through Image J software.
10
Fluorescent Oligonucleotide Tracking in Glioma Cells
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Because it is hard to label high-molecular-weight poly(I:C) with fluorescence, a fluorescein amidite (FAM)-bonded oligonucleotide was used as a surrogate. The FAM-bonded oligonucleotide (3ʹ-AAATTT-5ʹ-FAM) was designed and combined with Au@PP at a weight ratio of 0.05 (Au:FAM-oligonucleotide). GL261 mouse glioma cells were seeded in six-well plates at a density of 1.5×105 cells/well and then incubated with 20 μg/mL Au@PP/FAM or FAM-bonded oligonucleotide for 24 h. Next, the medium was removed and the cells were washed twice with phosphate-buffered saline (PBS). Fluorescence images were obtained by inverted fluorescence microscopy (Leica, Wetzlar, Germany).
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