Intra-cellular ROS levels were detected using dihydroethidium reagent. Frozen liver slices and L-02 cells were successively stained with dihydroethidium (D7008, Sigma, for liver slices; S0063, Beyotime Institute of Biotechnology, for cells) and DAPI (C1002, Beyotime Biotechnology). Specifically, the liver slices were stained with dihydroethidium for 30 min and DAPI for 10 min; the cells were washed with serum-free culture medium and incubated with the 5 μmol/L dihydroethidium at 37°C for 30 min and the DAPI for 10 min. The images were obtained under a fluorescence microscope (NIKON).
D7008
Manufactured by Merck Group
Sourced in United States
The D7008 is a laboratory equipment product offered by Merck Group. It is designed to provide precise and reliable performance for various laboratory applications. The core function of the D7008 is to facilitate efficient and accurate data collection and analysis, supporting the research and development needs of Merck's customers.
Automatically generated - may contain errors
Lab products found in correlation
A8056, by Merck Group (6 mentions)
D6883, by Merck Group (5 mentions)
Eclipse c1, by Nikon (5 mentions)
Lsm 780 meta confocal microscope, by Zeiss (4 mentions)
G1221, by Wuhan Servicebio Technology (4 mentions)
G1401, by Wuhan Servicebio Technology (4 mentions)
G1012, by Wuhan Servicebio Technology (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Cryostar nx50, by Thermo Fisher Scientific (2 mentions)
Eclipse ti sr, by Nikon (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
To pro 3 iodide, by Thermo Fisher Scientific (2 mentions)
Te2000, by Nikon (2 mentions)
Cryotome e, by Thermo Fisher Scientific (2 mentions)
Te 2000 fluorescent microscope, by Nikon (2 mentions)
To pro 3 iodide t3605, by Thermo Fisher Scientific (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
S0063, by Beyotime (1 mentions)
C1002, by Beyotime (1 mentions)
71 protocols using d7008
1
Quantification of Intracellular ROS Levels
2
Quantifying ROS Levels in Mouse Brains
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DHE staining was employed to detect the ROS levels in mouse brains as previously reported [34 (link)]. Fresh frozen brain Sects. (10 um) were incubated with 2 μmol/L fluorescent dye dihydroethidium (D7008, Sigma-Aldrich, Germany) at 37 °C for 30 min in a dark humidified chamber. After counterstaining with DAPI for 10 min, images of the ROS staining were captured via a fluorescence microscope (Leica, DMI8, Germany). For the quantitative analysis, the average of the DHE-positive cells was calculated from two randomly selected areas with the use of ImageJ software (ImageJ, USA) and expressed as a percentage of the total cell count.
3
Quantifying Cellular Oxidative Stress
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Detection of ROS was performed on frozen sections using dihydroethidium (D7008, Sigma-Aldrich, United States) which was diluted as 1:500 for 30 min at 37°C in a dark incubator. DAPI (4′, 6-diamidino-2-phenylindole, Beyotime, China) was added after the slides were dry and incubated for 10 min in the dark at room temperature. Slides were washed three times for 5 min each in phosphate buffered saline (pH 7.4) on a shaker. After the plate was blocked with an antifade mounting medium (Servicebio, China), the images were observed and acquired under a fluorescence microscope.
4
Quantification of ROS in Rat Brain
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DHE (D7008, Sigma-Aldrich, Shanghai), an oxidative fluorescent stain, was employed to assess ROS levels in rat brain tissue. Frozen brain tissue sections, 6-8 µm thick, were incubated with 50 µl of DHE (1:1000, 30 min) at 37°C for 30 min, shielded from light. Following this, the sections were treated with DAPI for 10 minutes. Subsequently, the sections were rinsed thrice with PBS. DHE staining images were captured using a light microscope, and the DHE-positive cells were quantified using ImageJ software.
5
Cryosectioning and ROS Visualization
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Fresh samples were embedded with OCT Compound (4,583, Sakura Finetek, Nagano, Japan) and sectioned with a Cryostat (Cryostar NX50, Thermo Fisher Scientific, Waltham, MA, United States). After rewarming at room temperature, the sections were incubated with ROS indicator dihydroethidium (DHE, D7008, SIGMA, St. Louis, MO, United States of America) at 37°C for 30 min in the dark, and then the nuclei were counterstained with DAPI for 10 min. Finally, inverted fluorescence microscope (IX73, Olympus, Tokyo, Japan) was used to observe.
6
Evaluation of Intracellular ROS Production
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Dihydroethidium (DHE, D7008, Sigma-Aldrich) was utilized to evaluate in situ production of ROS. Unfixed cryosections (10 μm) were stained with DHE (10 μmol/L) at 37 °C for 30 min in a humidified dark chamber. Ethidium fluorescence (excitation/emission at 488/610 nm) was examined by fluorescence microscopy (Eclipse Ti-SR, Nikon) at × 200 and evaluated by using Image-Pro Plus 6.0 version.
To assess the production of ROS by a second approach, isolated cardiomyocytes were prepared as described above and incubated with 2’,7’-dichlorofluorescein diacetate (DCFH-DA, S0033, Beyotime, China, 10 μmol/L) at 37 °C for 20 min. Mean fluorescence intensity (excitation/emission at 488/525 nm) which mirrored the levels of intracellular ROS was analyzed by flow cytometry (FACSCalibur, BD, USA).
To assess the production of ROS by a second approach, isolated cardiomyocytes were prepared as described above and incubated with 2’,7’-dichlorofluorescein diacetate (DCFH-DA, S0033, Beyotime, China, 10 μmol/L) at 37 °C for 20 min. Mean fluorescence intensity (excitation/emission at 488/525 nm) which mirrored the levels of intracellular ROS was analyzed by flow cytometry (FACSCalibur, BD, USA).
7
Flow Cytometric Analysis of Erythrocytes
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Twenty microliters of cell suspension was diluted in 80 μl of phosphate-buffered saline, stained with dihydroethidium (D7008; Sigma, Singapore) and SYBR green (10,000×; S9430; Sigma, Singapore) with 5 μg/ml and 5× as the final concentrations, respectively, and incubated for 20 min in the dark at room temperature as previously described (18 (link)). After staining, this suspension was analyzed using an Accuri C6 flow cytometer (BD Singapore). Forward scatter (FSC) and side scatter (SSC) signals of all erythrocytes were acquired via linear amplification, included potential abnormal cells due to hemoglobinopathies (22 (link), 23 (link)). Sixty thousand events were recorded, and flow cytometry analyses were done using FlowJo software (Tree Star). The proportion of schizont events for each sample was determined via a defined gating strategy (17 (link), 18 (link)).
8
Superoxide Quantification by DHE Assay
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Total superoxide (O2−) levels were assessed using dihydroethidium (DHE; D7008, Sigma-Aldrich). Samples were taken at the respective time point, incubated with DHE at a concentration of 2 µM for 30 min at 37 °C, washed 3× with PBS, transferred to SuperFrost Plus™ slides (Thermo Fisher Scientific, Waltham, MA, USA) by cytospin (80 rpm for 5 min at room temperature (RT); Shandon Cytospin II, GMI) and left to dry for 1 h at RT. After fixation in acetone for 10 min, the cells were rehydrated in PBS, counterstained using 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at RT, dehydrated in a graded series of ethanol (70%, 96% and 100%) and dried for 5 min before mounting with ProLong™ Gold Antifade Reagent (Thermo Fisher Scientific).
9
Nissl and DHE Staining of Rat Brain
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Rats were randomly selected, anaesthetized with chloral hydrate (350 mg/kg, ip), and perfused with PBS and then 4% paraformaldehyde. The whole brain was removed after perfusion, fixed, and preserved with 4% paraformaldehyde. The fixed brain tissue was sent to Servicebio (Wuhan, China) for paraffin sectioning.
Nissl staining was completed by Servicebio. The tissue sections were visualized with a light microscope and captured with Image Pro Plus. The number of normal cells and Nissl-stained cells was calculated using ImageJ Cell Counter. The percentage of neurodegeneration was used to evaluate nerve damage. The following formula was used: neurodegeneration percentage = pathological neuronal cells/total cells in the cortex or hippocampus in the field.
For DHE staining, after dewaxing and rehydration, tissue sections were incubated for antigen retrieval for 10 min and then incubated with dihydroethidium (100 μM, Molecular Probes, Sigma, D7008) at room temperature for four hours in the dark. The slides were rinsed three times with PBST, mounted with glycerin, and coverslipped. The tissue sections were visualized with a fluorescence microscope and captured with Image Pro Plus. The IOD was measured by Image Pro Plus.
Nissl staining was completed by Servicebio. The tissue sections were visualized with a light microscope and captured with Image Pro Plus. The number of normal cells and Nissl-stained cells was calculated using ImageJ Cell Counter. The percentage of neurodegeneration was used to evaluate nerve damage. The following formula was used: neurodegeneration percentage = pathological neuronal cells/total cells in the cortex or hippocampus in the field.
For DHE staining, after dewaxing and rehydration, tissue sections were incubated for antigen retrieval for 10 min and then incubated with dihydroethidium (100 μM, Molecular Probes, Sigma, D7008) at room temperature for four hours in the dark. The slides were rinsed three times with PBST, mounted with glycerin, and coverslipped. The tissue sections were visualized with a fluorescence microscope and captured with Image Pro Plus. The IOD was measured by Image Pro Plus.
10
Quantifying Reactive Oxygen Species via DHE
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Dihydroethidium (DHE), a commonly used indicator of ROS production is oxidized by ROS, forming ethidium that fluoresces red when intercalated with DNA. To determine the levels of ROS, muscle sections were incubated with DHE (D7008, Sigma-Aldrich, Saint Louis, MO, USA) for 30 min at 37 °C. In brief, 5 mM DHE was applied to the muscle sections and thereafter in situ fluorescence was assessed using fluorescence microscopy. DHE staining was quantified by measuring pixels exceeding a specified threshold, which was set in order to eliminate interference from any background fluorescence. The whole cross-section area was used for quantification and the percentage of area with positive staining was calculated.
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