For immunofluorescence analysis, T98G cells were grown on a glass coverslip, fixed with 4% paraformaldehyde (BioShop, Burlington, ON, Canada) in PBS, washed with PBS, and permeabilized with 0.1% Triton X-100, before addition of the appropriate primary and secondary antibodies. Microscopy was performed with a confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan), and the images were captured and processed using FV10-ASW 3.1 Viewer software (Olympus).
Fv10 asw 3.1 viewer software
Manufactured by Olympus
Sourced in Japan
The FV10-ASW 3.1 Viewer software is a tool designed for the visualization and analysis of microscope image data. It provides a user interface for viewing, navigating, and examining images captured with Olympus microscope systems.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview fv1000 confocal laser scanning microscope, by Olympus (2 mentions)
Fv1000, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole mounting solution, by Vector Laboratories (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Cy3 conjugated affinipure donkey anti goat igg h l, by Proteintech (1 mentions)
Fitc conjugated affinipure donkey anti rabbit igg h l, by Proteintech (1 mentions)
4 protocols using fv10 asw 3.1 viewer software
1
Immunofluorescence Analysis of T98G Cells
2
Immunofluorescence Imaging of NF-κB Activation
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Briefly, the cells were seeded on 15 mm microscope cover glass (Paul Marienfeld GmbH & Co., KG, Lauda-Königshofen, Germany) at a density of 5×106 cells/well in 12-well plates and grown overnight, followed by pre-treatment with essential oil and LPS stimulation for 24 h. For antibody labeling, the cells were first fixed with 37% formaldehyde and 95% ethanol (1:4) for 15 min at room temperature, followed by washing with 1X PBS three times (5 min/wash) and blocking with 1% BSA (Bioshop, Canada, Inc., Burlington, ON, Canada)/1X PBS for 1 h at room temperature. The cells were probed overnight with a 1:100 ratio of diluted antibody (p-p65) at 4°C. Following washing with 1X PBS four times (7-10 min/wash), the cells were blocked with 1:250 diluted anti-rabbit Alexa fluor 594 conjugate Red at room temperature for 1 h. The cells were then washed with 1X PBS and mounted with 4′,6-diamidino-2-phenylindole mounting solution on slides purchased from Vector Laboratories, Inc. (Burlingame, CA, USA) and were designated for confocal image analysis. All confocal images were captured with a ×20 oil objective (numerical aperture 3.5) lenses of Olympus Fluoview FV1000. FV10-ASW 3.1 viewer software (Olympus Corporation, Tokyo, Japan) was used to extract the images.
3
Immunohistochemical Analysis of TREM-1 in Sepsis Murine Model
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The cecal ligation and puncture (CLP)-induced sepsis was performed on three C57BL/6 mice as described before (22 (link)). The lung tissues of CLP mice and healthy mice were fixed with 4% paraformaldehyde, and tissue sections were subjected to immunohistochemistry analysis with mouse TREM-1 antibody antigen affinity-purified polyclonal goat IgG (R&D) and rabbit anti-beta actin polyclonal antibody (Proteintech), followed by FITC-conjugated affinipure donkey anti-rabbit IgG (H + L) (Proteintech) and CY3-conjugated affinipure donkey anti-goat IgG (H + L) (Proteintech). After washing five times, all sections were analyzed with a Fluoview™ Fv1000 laser scanning confocal microscope (OLYMPUS) and FV10-ASW3.1 viewer software (OLYMPUS). 405, 488, and 559 nm wavelengths were used, because the Excitation/Emission wavelengths of Hoechest 33258, FITC, and Cy3 dye were 405/461, 488/520, and 550/570 nm, respectively.
The UPLSAPO 100× (NA: 1.40) objective was used, and the zoom was ×1.0, so the magnification was 100 × 1. Imaging fields were chosen at random, and Z-sections were optimized for a number of cells. The image size of all images was 1,024 × 1,024 (pixels).
The UPLSAPO 100× (NA: 1.40) objective was used, and the zoom was ×1.0, so the magnification was 100 × 1. Imaging fields were chosen at random, and Z-sections were optimized for a number of cells. The image size of all images was 1,024 × 1,024 (pixels).
4
Fluorescent Imaging of Ca2+ Signaling
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Cells were seeded on a confocal dish with a glass bottom. The cells were loaded with dye by incubating with 5 μM Ca2+-sensitive probe Fluo 4-AM in the presence of 0.05% Pluronic F-127 in HBSS for 30 min at 37°C, and then washed three times with HBSS to remove the extracellular Fluo 4-AM and incubated in HBSS containing 1% FBS for 20 min at 37°C. Cells were treated initially with HEPES buffer and then with buffer containing 100 μM C2-ceramide. Changes in fluorescent intensity were monitored using an Olympus FluoView FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). Image was analyzed by Olympus FV10-ASW 3.1 Viewer software, using Time-series mode.
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