Cells (50 × 105 cells per well) were plated on glass coverslips in a six-well plate for 18 hours, fixed, and permeabilized using 4% paraformaldehyde (20 min) and 0.1% Triton X-100 in 1× PBS (pH 7.4) for 10 min. The cells were then blocked with 1% bovine serum albumin and dissolved in PBS (pH 7.4) for 1 hour. Cells were incubated for 18 hours at 4°C with primary antibodies in blocking solution followed by Alexa Fluor 488–, Alexa Fluor 594–, or Cy5-conjugated secondary antibodies (1:500) for 60 min. Immunofluorescence was performed using a Leica TSC SP2 AOBS TCS confocal or Olympus FV10i microscope with 543-nm and 488-nm channels for visualizing red and green fluorescence (50 (link)). Cilia were imaged using anti–Ac-tubulin antibody. Images were taken at 63× magnification. At least three random fields were selected for images. Images and overlays were analyzed in Leica LAS AF software (52 (link), 68 (link)).
Fv10i microscope
Manufactured by Olympus
Sourced in Japan, United States
The FV10i microscope is a compact and versatile inverted fluorescence microscope designed for advanced imaging applications. It features a high-performance optical system, a motorized stage, and a user-friendly software interface. The FV10i is capable of capturing high-resolution images and performing various imaging techniques, including confocal, multiphoton, and live-cell imaging.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 nlo quasar confocal multiphoton microscope, by Zeiss (3 mentions)
Fluoview software, by Olympus (2 mentions)
Streptavidin alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fluoview 1000, by Olympus (2 mentions)
Tsc sp2 aobs tcs confocal, by Olympus (1 mentions)
Las af software, by Leica (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Culture insert 2 well, by Ibidi (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Sulfo nhs lc biotin, by Apexbio (1 mentions)
Cm1900, by Leica (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
P35g 1.5 10 c, by MatTek (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ab18413, by Abcam (1 mentions)
Airyscan, by Olympus (1 mentions)
Metamorph digital imaging system, by Nikon (1 mentions)
Texas red dextran, by Thermo Fisher Scientific (1 mentions)
34 protocols using fv10i microscope
1
Immunofluorescence Imaging of Cilia
2
Myocardial and Cell Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Myocardial apoptosis and cell apoptosis were determined by an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) as previously described (22 (link)). In brief, at the end of the experiment, the myocardial tissues and H9c2 cells were fixed in 4% paraformaldehyde for at least 24 h. After the paraffin-imbedded sections were prepared, the manufacturer's instruction for TUNEL staining was followed. The apoptotic myocardial cells and H9c2 cells were stained with TUNEL staining solution, and nuclei were visualized by DAPI staining. Then the images were obtained with an Olympus FV10i microscope (Olympus, Tokyo, Japan); and an apoptotic rate was presented as the count of TUNEL-positive cardiomyocytes to the total number of cells.
3
Intracellular ROS Detection by DCFH-DA Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A non-fluorescent 2′,7′-dichlo rofluorescein-diacetate (DCFH-DA, 10 μM) intracellular probe was used to detect intracellular ROS formation. Approximately 1×103 cells/well were seeded into a 96-well plate. After 24 h, the cells were exposed to MHY4381 (1 μM) or SAHA (1 μM) for 12 h followed by incubation with 10 μM DCFH-DA in serum-free media for 30 min at 37°C. The cells were washed twice with PBS, and DCF fluorescence was detected using a fluorometric imaging plate reader at excitation and emission wavelengths of 488 nm and 520 nm, respectively. To observe the fluorescence image, the cells were seeded into a confocal dish and treated as described previously with the given compounds (MHY4381 or SAHA, each 1 μM) and DCFH-DA in serum-free media for 30 min at 37°C. After washing the cells with PBS, their images were captured using an FV10i microscope (Olympus).
4
Visualizing Apoptotic Nuclear Changes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Morphological changes in the nuclear chromatin in apoptotic cells were assessed by DAPI staining. Cells (1×104) were seeded into a 6-well plate dish and treated with MHY4381 (0.1, 0.5, or 1 μM) or SAHA (1 μM) for 48 h, followed by fixation with methanol and staining with DAPI solution (1 μg/mL). The staining solution was discarded, and the cells were washed with cold PBS. Confocal microscopy was performed using an FV10i microscope (40x, Olympus, Tokyo, Japan) to visualize apoptotic cells.
5
Directed Migration Assay for HaCaT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HaCaT keratinocytes stably expressing shRNA targeting human COL17A1 and SCR were previously established (Liu et al., 2019 (link)) and were maintained in DMEM with 10% FBS. For the directed migration assay, these HaCaT cell lines were prepared at 0.8 × 106 cells/ml in DMEM with 10% FBS containing 500 ng/ml Dox (Wako; 04931121), and a 70-µl volume of the suspension was seeded into each well of a Culture-Insert 2 well (Ibidi; 81176) on a 35-mm cell culture dish (Grainer Bio-one; 627965) and incubated overnight. After treatment with and without 0.4 µg/ml mitomycin C (Kyowa Kirin) for 2 h and further incubation with DMEM with 10% FBS containing 500 ng/ml Dox, the culture insert was removed from the cell culture dish, and serial images were obtained at 30-min intervals for 24 h. Time-lapse images were acquired with an FV10i microscope (Olympus) and Fluoview software (Olympus).
6
Cell Cycle and Locomotion Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Time-lapse images were acquired with an FV10i microscope (Olympus) and Fluoview software (Olympus) and analyzed with Fiji (Schindelin et al., 2012 (link)). Cell cycle duration was defined as the duration between two rounds of cell division. Cell locomotion speed was defined as the amount of change in the position of the cell nucleus. The position of the nucleus in the cell was given as the center of the circle that approximated the nuclear shape. The rotational speed of a two-cell colony was assessed as the amount of change in the angle between cells in a two-cell colony. The angle of contact surface between cells in a two-cell colony was given by the top-right angle of the contact surface against the perpendicular axis.
7
Visualizing Lipid Droplets in Ascocarp
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal microscopy was performed to visualize lipid droplets within the ascocarp. Samples were sliced with a surgical scalpel and stained with 10.0 μg mL -1 BODIPY 493/503 (Thermo Fisher Scientific, Pittsburgh, USA) in a phosphate-buffered saline (PBS) buffer for 2 days at 23 C. After two washes with a PBS buffer, the samples were then observed using an Olympus FV10i microscope (Olympus Scientific Solutions Americas, Waltham, Massachusetts). An argon (488 nm) laser was used for BODIPY (emission: 510-530 nm).
8
Labeling Liver and Brain Tissues with Biotin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mice were deeply anesthetized with 2% sodium pentobarbital, and cardiac perfusion was performed with phosphate-buffered saline (PBS) for 20 min. Then the mice were transcardially perfused with Sulfo-NHS-LC-Biotin ( ) (A8003; APExBIO) dissolved in PBS. The liver and brain tissues were collected and immersed in 4% paraformaldehyde for 4 h, followed by a solution of 30% sucrose overnight. The dehydrated liver and brain tissues were stored at . The tissues were embedded in OCT and cut into slides using a vibratome (CM1900; Leica). The frozen slices were dried at room temperature for 15 min. Slides were immersed in PBS for 10 min to remove the OCT. Triton (0.3%) was added to the glass slide for drilling for 10 min. Then the slides were washed with PBS for 10 min and sealed with a 3% bovine serum albumin (BSA) solution at 37°C for 30 min. The frozen sections were stained with streptavidin–Alexa Fluor 594 (Invitrogen) at 37°C for 1 h, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) at 37°C for 15 min. The images were captured using a confocal fluorescence microscope FV10i microscope (Olympus).
9
Visualization of Actin Dynamics in Osteoclasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW 264.7 cells were plated on a 35-mm glass-bottomed culture dish (P35G-1.5-10-C, MatTek, Ashland, MA; 3910-035-NYP, Iwaki, Asahi Glass, Tokyo, Japan), and transfected with EGFP-actin (6116-1, Clontech, CA) using FuGENE HD transfection reagent (E231A, Promega, Madison, WI) according to the manufacturer's instructions. The cells were then cultured in the presence of sRANKL for 4 days. Immediately before imaging, the culture medium was again supplemented with 100 ng/ml sRANKL. Time-lapse confocal images of EGFP-actin in osteoclasts were acquired with an Olympus FV10i microscope (Olympus, Tokyo, Japan) equipped with a 60× water objective lens in a 5% CO2 humidified atmosphere at 37°C, every 4 s for at least 13 min. Imaging at 1 s intervals yielded similar results. The focal width of the confocal scanning was set to 2.0 µm. Kymographs were generated from the time series images using ImageJ (https://imagej.nih.gov/ij/ ). The actin flow rate was calculated by identifying the trajectory of the EGFP-positive spots on the kymograph, as shown in Fig. 1 A. Data were collected from 17 independent experiments. For the inhibitor (CK-666) experiments, recording was performed on a Nikon A1si microscope using a 60×/1.4 NA oil objective lens at room temperature. The cells were imaged for 10 min before the addition of CK-666 and for 40 min thereafter.
10
Immunofluorescence Localization of Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells cultured on gelatin-coated glass coverslips were fixed with 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100. After blocking non-specific binding sites with 5% BSA, 0.1% Tween 20 in PBS for 1 h at room temperature, we incubated the fixed cells (1 μg/ml) or cryosections of testis (5 μg/ml) with 20B2 primary antibody overnight at 4 °C. We stained cells with AlexaFluor 488-conjugated goat anti-mouse IgG (Invitrogen) for 1 h at room temperature, followed by counterstaining with DAPI (1 μg/ml, Dojindo, Kumamoto, Japan) to visualize the nucleus and acquired confocal fluorescence images with an FV10i microscope (Olympus). As negative control, Mouse control IgG2a (1 μg/ml for fixed cells, 5 μg/ml for cryosections, Abcam, ab18413) were used. For absorbance experiments, the 20B2 antibody was pre-incubated with GP (1 μg/ml for fixed cells, 5 μg/ml for cryosections).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!