After 60%–70% cellular confluency was achieved in 60 mm dishes under standard culture conditions, cells were treated with 10 µL of demecolcine (stock: 10 µg/mL) for 3–4 h. Cells were harvested using trypsin (Corning™ 25053CI) and the cell pellet was gently treated with hypotonic solution (75 mM KCl) for 40–60 min at 37 °C and fixed in cold methanol/acetic acid (3:1). Two or three drops of suspended cells were applied to glass slides and chromosomes were stained with DAPI and counted using confocal microscopy (Olympus Fluoview FV1200 Confocal Laser Scanning Microscope, Olympus, Japan).
Fluoview fv1200 confocal laser scanning microscope
Manufactured by Olympus
Sourced in Japan, United States
The FLUOVIEW FV1200 is a confocal laser scanning microscope designed for high-resolution fluorescence imaging. It utilizes laser excitation and a pinhole aperture to capture sharp, in-focus images by rejecting out-of-focus light. The FV1200 is capable of capturing detailed, three-dimensional images of fluorescently-labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Fluorescein dextran solution, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Ix53 inverted fluorescent microscope, by Olympus (2 mentions)
Cellsens software, by Olympus (2 mentions)
α sma, by Abcam (1 mentions)
Collagen 1, by Affinity Biosciences (1 mentions)
Collagen 1, by Abcam (1 mentions)
Slowfade diamond, by Thermo Fisher Scientific (1 mentions)
Phalloidin alexa 568, by Thermo Fisher Scientific (1 mentions)
Ab32570, by Abcam (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
S2532, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Fv10 asw ver4.1, by Olympus (1 mentions)
Alexa fluor 488 labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti α sma primary antibody, by Merck Group (1 mentions)
Lionheart fx automated microscope, by Agilent Technologies (1 mentions)
Superclonal recombinant secondary antibody, by Thermo Fisher Scientific (1 mentions)
29 protocols using fluoview fv1200 confocal laser scanning microscope
1
Chromosome Counting by Confocal Microscopy
2
Immunofluorescence analysis of IMR90 cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IMR90 cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 20 min and then blocked with 1% BSA for 30 min. Cells were incubated with α-SMA and Collagen I were visualized with an overnight with specific fluorochrome primary antibodies including α-SMA (Abcam, United States), Collagen I (Affinity, China) at a concentration of 1:100. After extensive washing with PBS, cells were incubated with goat Alexa Fluor 488-labeled secondary antibody (Life Technologies, United States) for 1 h at room temperature and nuclei were stained with DAPI. The images were obtained by using Olympus FluoView® FV1200 confocal laser scanning microscope (Olympus Corporation, Center Valley, PA).
3
Karyotype analysis of cell lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 60%–70% of confluency was achieved in 60 mm dishes, cells were treated with 10 μL of demecolcine (10 μg/mL) for 3–4 h. Cells were harvested using trypsin (Corning™ 25053CI) and the pellet was gently treated with hypotonic solution (75 mM KCl) for 40–60 min at 37 °C and fixed in cold methanol/acetic acid (3:1). Two or three drops of suspended cells were applied to glass slides and chromosomes were stained with DAPI and imaged by confocal microscopy (Olympus Fluoview FV1200 Confocal Laser Scanning Microscope, Olympus Australia Pty. Ltd., Melbourne, Victoria, Australia). Counting was performed manually in ImageJ. Karyotype assessment via G-band analysis was performed by a commercial genotyping service (StemCore Facility, Brisbane, QLD, Australia) and 50 metaphases were analysed per cell line.
4
Immunofluorescence Imaging of Sec12 and Sec23
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
INS-1 832/13 cells cultured on coverslips were fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized, and blocked with 0.1% Triton X-100 in PBS containing 5% FBS and 0.3% bovine serum albumin (BSA) for 1 h. For Sec12 and Sec23 staining, cells were fixed in pre-cooled (−20 °C) methanol for 6 min, permeabilized and blocked with 0.1% Triton X-100 in PBS containing 5% BSA for 30 min, incubated with primary antibodies for 60 min, washed three times with PBS, and further incubated with Alexa Fluor 488-, 555-, or 594- conjugated secondary antibodies (1:1000 dilution; Invitrogen) for 60 min. The samples were washed five times, mounted using Slow Fade Diamond (Invitrogen) containing DAPI (157574; Molecular Probes, Eugene, OR, USA), and visualized using a Fluoview FV1200 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) equipped with a 60× oil immersion objective lens (1.35 NA).
5
Quantifying Fluorescence Recovery After Photobleaching
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transfected with EGFP the day before the experiment and plated on laminin. Photobleaching was performed using an Olympus FluoView FV1200 Confocal Laser Scanning Microscope with 70% 405 nm laser on an ROI comprising most of the cytoplasm of the cell using the laser light stimulation (SIM) scanner with a 60X objective (UPLSAPO60XS, Numerical Aperture 1.3). Images were acquired at full speed (every 1.1s). The mean GFP intensity in a ROI of fixed size was measured on the images. To quantify the amplitude of fluorescence loss following photobleaching, we measured the minimum intensity reached in a sister cell still connected to the photobleached cell by a bridge, or in an unconnected neighbor (Figure 4 D). Fluorescence levels were normalized to the initial fluorescence levels, to take into account cell-to-cell variability in GFP expression.
6
Perfusability Assessment of Tumor Microvasculature
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm the perfusability of MVNs in tumor devices, the culture medium in one media channel was aspirated, followed by injection of 100 μL of 10 μg mL−1 10 kDa MW fluorescein dextran solution (Invitrogen). The process was then repeated for the other media channel. The device was imaged within 5 min under a confocal microscope. For confocal imaging, an Olympus FLUOVIEW FV1200 confocal laser scanning microscope with a 10× objective was used. Z-stack images were acquired with a 5 μm step size. All images shown are collapsed Z-stacks, displayed using range-adjusted Imaris software, unless otherwise specified. Vessel percentages in the tumor region and tumor nearby 50 μm distance region were measured using ImageJ (NIH, U.S., demonstrated in Figure S8 , Supporting Information ). FB area was also quantified using ImageJ (NIH, U.S.).
7
Fixation and Staining of Cellular Blebs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples, isolated blebs and cells were spun on poly-L-lysine coated 25 mm coverslips by centrifugation at 460 g for 10 min. Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min followed by 10 min permeabilisation with 0.2% Triton X-100 at room temperature. Blebs were fixed with combined permeabilisation-fixation for 6 min with 4% PFA in intracellular buffer with 0.2% Triton X-100 followed by 14 min fixation with 4% PFA in intracellular buffer at room temperature, followed by three washes with PBS. Samples were stained with DAPI and phalloidin–Alexa568 (1:500 dilution, Thermo Fisher Scientific, A12380) for 1 h, followed by three washes with PBS.
Samples were imaged using Olympus FluoView FV1200 Confocal Laser Scanning Microscope using a 60× oil objective (NA 1.4).
Samples were imaged using Olympus FluoView FV1200 Confocal Laser Scanning Microscope using a 60× oil objective (NA 1.4).
8
Perfusability Assessment of Tumor Microvasculature
9
Immunostaining of Brain Microvascular Cells
10
Endoxifen and Curcumin Effects on Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Coverslip was coated using a poly-L-lysine. Coverslips were placed in each well of a six well cell culture plate. Cells (104) were seeded into each well of a 6-well plate. After 72-hour incubation to allow cells to adhere, cells were treated with endoxifen+β-estradiol with or without curcumin or DMSO every 48 hours until the 60% of cell confluency. Coverslips were washed and mounted on glass slide. All images were captured on Olympus Fluoview FV1,200 Confocal Laser Scanning Microscope (Olympus, Japan) using a 40× lens. Images were captured using FV10-ASW ver 4.1 and Cellsens ver 1.14 imaging software (Olympus, Japan).
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!