Antibodies were incubated with cells seeded in 8-well culture chamber slides (Fisher Scientific) for the indicated amount of time. To assess pathway of internalization (macropinocytosis), cells were co-incubated with TexasRed-conjugated 70-kDa neutral dextran (ND70-TR, Life Technologies), a marker for macropinocytosis (16 (link)). Post incubation cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS/1% FBS/0.2% Triton-X100. Cell-associated antibodies were stained with Alexa Fluor® 488- or 647-labeled goat anti-human IgG (Jackson ImmunoResearch) for 1 hour at room temperature. Lysosomes were detected by rabbit anti-lysosomal-associated membrane protein 1 (LAMP1) antibody (Cell Signaling) followed by incubation with Alexa Fluor® 647-labeled goat anti-rabbit IgG (Jackson ImmunoResearch). For analysis of antibody localization in tumor spheres, spheres were collected by centrifugation at 500 × g for 5 min, washed, fixed, permeabilized, and immunolabeled using antibodies described above. CyGEL™ (Abcam) was used to immobilize spheres in 8-well chamber slide for microscope analysis. For imaging, cells or spheres were counterstained using Hoechst33342 (Thermo Scientific) and imaged by FluoView® FV10i laser confocal microscope (Olympus) with an Olympus 60× phase contrast water-immersion objective.
Fluoview fv10i laser confocal microscope
Manufactured by Olympus
Sourced in Japan
The FluoView® FV10i is a laser confocal microscope designed for high-resolution imaging of fluorescently labeled samples. It utilizes a laser as the illumination source and a pinhole to eliminate out-of-focus light, providing optical sectioning capabilities for 3D imaging. The FV10i is capable of capturing detailed images with minimal background noise, making it a versatile tool for a variety of life science applications.
Automatically generated - may contain errors
4 protocols using fluoview fv10i laser confocal microscope
1
Antibody Internalization and Lysosomal Trafficking
2
Detecting SIAH1-CPSF1 Interaction by PLA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SIAH1-CPSF1 interaction was detected by PLA using anti-SIAH1 and anti-CPSF1 antibodies and PLA probes in 22Rv1 cells. The experiments were carried out as previously mentioned.43 (link) In brief, 22Rv1 cells were grown on glass coverslips, fixed with methanol or 4% PFA, permeabilized with acetone or 0.2% PBS-Tween, and then incubated with SIAH1 and CPSF1 antibodies (Olink Biosciences, Uppsala, Sweden) at 4°C overnight. PLA minus and PLA plus probes were added and incubated for 1 h at 37°C. Further oligonucleotides were added, and allowed to hybridize to the PLA probes, and a ligase joined the two hybridized oligonucleotides. The fluorescence images were captured using an Olympus Fluoview FV10i Laser confocal microscope (Olympus, Tokyo, Japan). Images were collected sequentially on a confocal laser scanning microscope (Olympus UPLSAPO 60XO, NA 1.35) and analyzed using Olympus FV10-ASW version 3.0 Software.
3
Antibody-Antigen Colocalization and Nuclear Translocation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at 1.5 × 104/well on Labteck 8-well culture chamber slides (Thermo Fisher Scientific). To evaluate antibody-target antigen (LRP6) colocalization, cells were incubated with each antibody for 1 h in growth media, fixed with 4% PFA (paraformaldehyde), permeabilized in PBS/1% FBS/0.2% Triton-X100, and further incubated with FITC-labeled goat anti-human IgG for antibody detection and Alexa Fluor 647®-labeled anti-LRP6 IgG for LRP6 staining. To study nuclear β-catenin translocation, cells were seeded in chamber wells as described above, incubated with the testing antibody in DMEM/10% FBS supplemented with 20% Wnt3a conditioned medium (Wnt3a-CM) for 24 h, stained with mouse anti-β-catenin primary antibody (Cell Signaling Technology) for β-catenin detection, followed by Alexa Fluor® 647-labeled rabbit anti-mouse secondary IgG (Jackson ImmunoResearch). All cells were counterstained using Hoechst33342 (Thermo Scientific) and imaged on FluoView® FV10i laser confocal microscope (Olympus) with an Olympus 60X phase contrast water-immersion objective.
4
Proximity Ligation Analysis of Phosphorylated STAT6 in Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proximity ligation assay was used to investigate the proximity of the epitopes recognized by the anti-STAT6 and anti-Tyrosine phosphorylation-specific antibodies, which represent the expression of phosphorylated STAT6 in cancer cells. The experiment was performed according to the manufacturer’s instructions. Briefly, after incubation with primary antibodies, the corresponding DuoLink® In Situ PLA probes (OLINK Bioscience, Uppsala, Sweden) were applied for 1 h at 37 °C as recommended. Subsequent ligations and detections using the DuoLink® In Situ Detection Reagents Red Kit (OLINK Bioscience, Uppsala, Sweden) were performed. Blocking, antibody hybridization, proximity ligation, and detection were performed according to the manufacturer’s recommendations. The fluorescence images were captured using an Olympus FluoView FV10i Laser confocal microscope (Olympus Corporation, Tokyo, Japan). Images were collected sequentially on a confocal laser scanning microscope (Olympus UPLSAPO 60XO, NA 1.35) and analyzed by Olympus FV10-ASW Version 3.0 Software. The results were quantified using MetaMorph® Microscopy Automation & Image Analysis Software.
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!