A cell climbing slice was prepared in a 24 well plate. When cells were attached to the wall, they were divided into a control group and salvianolic acid probe group. The cells were rinsed for 5 min with PBS buffer 3 times and then fixed in 3.7% formaldehyde at −4 °C for 10 min. The serum was diluted to 5% with room temperature PBS and incubated at room temperature for 30 min (closed liquid containing 0.1% Triton X-100). Primary antibodies used included rabbit anti-transgelin (dilution, 1:200; Abcam) and mouse anti-actin (dilution, 1:100; CST). After being rinsed gently with PBS three times, the cells were incubated at 37 °C for 1 h with Alexa Fluor® 488-conjugated goat anti-rabbit IgG (dilution, 1:150; KeyGEN BioTECH) and Alexa Fluor® 568-conjugated goat anti-mouse IgG (dilution, 1:250; Invitrogen) as secondary antibodies. The salvianolic acid probe was connected to 647 dyes by click reaction. The cells were viewed through a confocal microscopy system (Nikon, Japan).
Confocal microscopy system
Manufactured by Nikon
Sourced in Japan
The Nikon Confocal Microscopy System is a high-resolution imaging solution that utilizes a focused laser beam to scan and capture images of samples. The system provides optical sectioning capabilities, allowing users to obtain detailed, three-dimensional information about the internal structure of specimens. The core function of the Confocal Microscopy System is to enable the acquisition of high-quality, high-contrast images with improved resolution and depth of field compared to traditional widefield microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
N sim analysis software, by Nikon (2 mentions)
Vw h2ma, by Keyence (2 mentions)
Bz x700 all in one fluorescence microscope, by Keyence (2 mentions)
Bz x analyzer, by Keyence (2 mentions)
Rabbit anti transgelin, by Abcam (1 mentions)
Alexa fluor 568 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Keygen Biotech (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Nis elements ar 4, by Nikon (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
7 protocols using confocal microscopy system
1
Immunofluorescence Assay for Salvianolic Acid
2
Assessing Cochlear Oxidative Stress
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We characterized the level of oxidative stress in cochlear samples, by analyzing reactive oxygen species (ROS) amount through a dihydroethidium (DHE) assay. DHE is a lipophilic cell-permeable dye that, in the presence of free radicals, is rapidly oxidized to ethidium. The produced ethidium is fixed by intercalation into nDNA, so that it gives an indication of oxidative stress status.
Cochlear specimens (4 cochleae/group) were then incubated with 1 μM DHE (Invitrogen, Carlsbad, CA, USA, Cat. No. D23107) in PBS for 30 min at 37°C and then coverslipped with an antifade medium (FluorSave™ Reagent). Images were obtained by using two-photon excitation (792 nm, <140 fs, 90 MHz) performed using an ultrafast tunable mode-locked titanium:sapphire laser (Chameleon, Coherent) and a confocal microscopy system (Nikon).
Cochlear specimens (4 cochleae/group) were then incubated with 1 μM DHE (Invitrogen, Carlsbad, CA, USA, Cat. No. D23107) in PBS for 30 min at 37°C and then coverslipped with an antifade medium (FluorSave™ Reagent). Images were obtained by using two-photon excitation (792 nm, <140 fs, 90 MHz) performed using an ultrafast tunable mode-locked titanium:sapphire laser (Chameleon, Coherent) and a confocal microscopy system (Nikon).
3
Imaging and Analyzing Mitochondria in sIBM Cells
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To obtain images of mitochondria, cells from sIBM patients were stained with MitoTracker red (a molecular probe, shown in red), and the nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, shown in blue). The samples were then placed into a confocal microscopy system (Nikon, Tokyo, Japan). Images were acquired and analyzed using NIS-Elements with N-SIM analysis software (Nikon) and ImageJ. Briefly, the MitoTracker red signal in the images was changed to grayscale, and a suitable threshold level that allowed the signal intensity of the mitochondria to be distinguished from the background noise was set in ImageJ software. The lengths of the major and minor axes of the mitochondria were measured using ImageJ. Electron microscopy analysis was performed as previously reported [12 (link), 16 (link)]. For live-cell imaging, culture dishes with myoblasts and fibroblasts from sIBM patients stained with MitoTracker Green were placed into a KEYENCE BZ-X700 All-in-one Fluorescence Microscope (KEYENCE, Osaka, Japan). Images were taken for 5 min and converted to movie files using a BZ-X Analyzer (KEYENCE). The movies were analyzed with the video editing analysis software VW-H2MA (KEYENCE) to evaluate cell migration [17 (link)].
4
Mitochondrial Imaging in sIBM Cells
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To obtain images of mitochondria, cells from sIBM patients were stained with MitoTracker red (a molecular probe, shown in red), and the nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, shown in blue). The samples were then placed into a confocal microscopy system (Nikon, Tokyo, Japan). Images were acquired and analyzed using NIS-Elements with N-SIM analysis software (Nikon) and ImageJ.
Briefly, the MitoTracker red signal in the images was changed to grayscale, and a suitable threshold level that allowed the signal intensity of the mitochondria to be distinguished from the background noise was set in ImageJ software. The lengths of the major and minor axes of the mitochondria were measured using ImageJ. Electron microscopy analysis was performed as previously reported [12, 16] . For live-cell imaging, culture dishes with myoblasts and fibroblasts from sIBM patients stained with MitoTracker Green were placed into a KEYENCE BZ-X700 All-in-one Fluorescence Microscope (KEYENCE, Osaka, Japan). Images were taken for 5 min and converted to movie files using a BZ-X Analyzer (KEYENCE). The movies were analyzed with the video editing analysis software VW-H2MA (KEYENCE) to evaluate cell migration [17] .
Briefly, the MitoTracker red signal in the images was changed to grayscale, and a suitable threshold level that allowed the signal intensity of the mitochondria to be distinguished from the background noise was set in ImageJ software. The lengths of the major and minor axes of the mitochondria were measured using ImageJ. Electron microscopy analysis was performed as previously reported [12, 16] . For live-cell imaging, culture dishes with myoblasts and fibroblasts from sIBM patients stained with MitoTracker Green were placed into a KEYENCE BZ-X700 All-in-one Fluorescence Microscope (KEYENCE, Osaka, Japan). Images were taken for 5 min and converted to movie files using a BZ-X Analyzer (KEYENCE). The movies were analyzed with the video editing analysis software VW-H2MA (KEYENCE) to evaluate cell migration [17] .
5
Immunofluorescence Staining of RGS4
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Tissue samples on slides were deparaffinized and rehydrated using standard techniques. Sections were immersed in 3% H2O2 for 10 min to quench endogenous peroxidase activity. They were then treated with goat serum blocking reagent and incubated with anti-RGS4 antibodies for 1 h at room temperature. Samples were rinsed four times with phosphate-buffered saline (PBS) and then incubated with Alexa Fluor 488-labeled secondary antibodies (1:1000 dilution in 5% normal goat serum) for 1 h at room temperature. Samples were rinsed twice with PBS, and then glass coverslips were mounted on the slides using VECTASHIELD with DAPI (Vector Laboratories, Burlingame, CA). The tissue samples were analyzed and images were captured using a Nikon confocal microscopy system (Nikon, Tokyo, Japan).
6
Fluorescence Recovery After Photobleaching
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Fluorescence recovery after photobleaching (FRAP) assay was conducted using the FRAP module of the Nikon confocal microscopy system. The HBB–eGFP was bleached using a 488-nm laser beam. Bleaching was focused on a circular region of interest using 100% laser power and timelapse images were collected. Fluorescence intensity was measured using Nikon confocal microscopy system (NIS Elements AR 4.50.00). Values are reported as ratios relative to pre-bleaching time points. GraphPad Prism is used to plot and analyse the FRAP results.
7
Mitochondrial Dynamics in HK2 Cells
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HK2 cells were plated on PDL-coated glass coverslips in six-well tissue culture plates. After 24 hours, the cells were transfected with Dsred-Mito plasmid. After 48 hours, the slides were rinsed with PBS and fixed for 10 minutes with 4% paraformaldehyde in PBS at room temperature. This was followed by permeabilization with 0.05% Triton X-100 in PBS for 15 minutes. After rinsing twice with PBS, the slides were incubated with Cytochrome c antibodies for 1 hour at room temperature, rinsed four times with PBS and incubated with Alex 488 labeled secondary antibodies (1:1000 in 5% normal goat serum) for 1 hour at room temperature. After rinsing twice with PBS, slides were finally mounted on glass cover slips using VECTASHIELD (Vector Laboratories), and images were collected and analyzed on a Nikon confocal microscopy system.
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