Cellular fractions of vaginal lavage fluid from mice at 2 days postinoculation were pelleted by centrifugation and washed in sterile water to eliminate PMNs by hypotonic lysis. The remaining cells were washed in PBS and incubated with calcofluor white (Fluka) (1 mg/ml) (C. albicans cell wall), SYTO 9 green fluorescent nucleic acid stain (Invitrogen) (2.5 µM) (live cells), and propidium iodide (Invitrogen) (30 µM) (dead cells) for 20 min at room temperature in the dark. Following incubation, 10 µl of cell suspension was placed on a glass slide and examined using an Olympus FV100 confocal microscope with FluoView software.
Fv100 confocal microscope
Manufactured by Olympus
Sourced in Japan
The FV100 is a confocal microscope designed for advanced imaging applications. It features a high-resolution optical system and advanced imaging capabilities to capture detailed images of samples. The FV100 is a versatile instrument suitable for a range of research and analysis tasks.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield plus antifade, by Vector Laboratories (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Syto 9 green fluorescent nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Rabbit monoclonal anti γh2a x antibody, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Vivaview lcv110u, by Olympus (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Formalin, by Merck Group (1 mentions)
Celltracker orange cmra dye, by Thermo Fisher Scientific (1 mentions)
Transwell insert, by Corning (1 mentions)
Dapi solution, by Solarbio (1 mentions)
Alexa fluor 488 conjugate, by Cell Signaling Technology (1 mentions)
15 protocols using fv100 confocal microscope
1
Visualizing Fungal Infection in Mice
2
Visualizing Autophagosome Formation Dynamics
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EBV-B cells were transfected with a plasmid coding for LC3 fused to enhanced green fluorescent protein (eGFP), kind gift of John Brumell (University of Toronto), using a microporator (Digital Bio) with 100 μL Neon kit (Life Technologies). The next day, GFP+ cells were sorted by flow cytometry using a BD FACSAria III (BD Biosciences). Sorted cells were incubated with PapMV-AF647 (10 μg/mL) or rapamycin (0.1 μM) for 3 hours. Cells were cytospun using a Cytofuge 2 (StatSpin) on slides and coverslips were mounted with ProLong Gold Antifade Mountant with DAPI (4′,6-diamidino-2-phenylindole, Life Technologies). Pictures were taken on an Olympus FV100 confocal microscope and analyzed with ImageJ software for LC3-GFP+ vesicle count and surface quantification.
3
Immunofluorescent Localization of eNOS and Caveolin-1
4
Detecting DNA Damage via γH2AX Foci
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Cells were plated in 30-mm dishes and cultured for 72 h at 37°C. To detect irradiation-induced DNA double-strand breaks (DSBs), cells were treated with a 2-Gy dose of irradiation from an external X-ray source (RAD SOURCE) at room temperature and incubated for 0.5 and 24 h. Unirradiated cells served as controls. To detect H2AX phosphorylation, cells were sequentially fixed in 4% formaldehyde (Sigma–Aldrich) for 15 min and 50% methanol in PBS for 10 min. The cells were subsequently blocked with 5% bovine serum albumin for 30 min, incubated with a rabbit monoclonal anti-γH2AX antibody (1:1,000, Cell Signaling Technology, Boston, USA) for 30 min, washed in PBS, incubated with an Alexa 488-conjugated (Molecular Probes, USA) secondary antibody for 30 min, and counterstained with DAPI (Invitrogen). Images were captured using an Olympus FV100 confocal microscope. γH2AX-positive cells were defined as those with more than 20 γH2AX foci. Five random fields per coverslip were selected to calculate the number of γH2AX-positive cells. Assays were performed in triplicate to eliminate intra-assay variability.
5
Dual Fluorescence Immunolabeling Protocol
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The first part of our protocol for double-labeling was similar to the methodology described above, with the exception of the endogenous peroxidase quenching step, which was omitted as the antibodies would be fluorescently tagged. After incubation with the first primary antibody (T-Antigen, mouse monoclonal), an Alexa Flour 488-conjugated anti-mouse secondary antibody was incubated for 1 h in the dark. Sections were then washed thoroughly with PBS, blocked again, and a second primary antibody raised in a different species than the first one (LMP, rabbit monoclonal) was incubated overnight. Finally, a second Alexa Fluor 568-conjugated anti-rabbit secondary antibody was incubated for 1 h in the dark, and slides were cover-slipped with an aqueous mounting media containing DAPI (Vectashield Plus Antifade, Vector Laboratories) and visualized in an Olympus FV100 confocal microscope.
6
Visualizing Pollen Tube Dynamics with Fluorescence Microscopy
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The pollen tubes of the homozygous pCAMBIA1300-Lat52-GFP, pCAMBIA1300-Lat52-Ann5G26EG28E-GFP and pCAMBIA1300-Lat52-Ann5G257EG259E-GFP lines were observed using an Olympus FV100 confocal microscope with a 100× oil objective. GFP was excited using a 488-nm argon laser, and emission was detected through 525±5.5 nm filters. Serial confocal optical sections were taken at a step size of 0.5 µm, and two Kalman-filtered scans were averaged for each optical section.
7
Microangiography and Phagocytosis Assay
8
Visualizing Cell Engulfment Dynamics
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Video 1 was reconstructed from images taken on an Olympus FV100 confocal microscope using a Plan Apochromat N 60× oil objective. Videos 2, 6, 7, 9, and 10 were imaged on an Olympus Viva-view LCV110U using a 10× objective for 2, 6, 9, and 10 and a 20× objective for 7. Videos 3, 4, and 5 were imaged on a Carl Zeiss cell observer inverted microscope with an LD Plan-Neofluar 20× objective. Video 8 was reconstructed using NIS-Elements software from images taken on a Nikon A1rSi laser point scanning confocal microscope with a Plan Apochromat λ 60× oil objective. All videos began 6–7 d after doxorubicin treatment, unless otherwise noted. Videos were created of senescent 4226 cells engulfing NT cells in seven independent experiments, totaling 199 separate videos. Representative videos of complete cell engulfment captured are shown. Videos were created of senescent MCF-7 cells engulfing NT cells in three separate experiments. One experiment was captured on an IncuCyte and two other experiments were captured on other microscopes mentioned above, totaling 35 videos.
9
Immunofluorescence Staining of Rat PASMCs
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Primary cultured rat PASMCs were fixed in 4% paraformaldehyde at +4°C for 10 min. Cells were then washed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS for 10 min. Unspecific binding was reduced by blocking with 5% BSA (Sigma) in PBS for 60 min. Specimens were then incubated with primary antibody ACTA2 (Abcam, Cat.#: ab220179) at +4°C overnight, then with Alexa Flour 488 (anti-mouse)-conjugated secondary antibody at room temperature for 2 h. Nuclei were stained with DAPI. Fluorescent images were acquired using an Olympus FV100 confocal microscope.
10
Double Immunofluorescence Labeling Protocol
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The first part of our protocol for double labeling is similar to the methodology described above, with the exception of endogenous peroxidase quenching step, which was not performed. However, after incubation with the first primary antibody (T-Antigen), an Alexa Flour 488-conjugated anti-mouse secondary antibody was incubated for 1 h in the dark. Sections were then washed thoroughly with PBS, blocked again, and a second primary antibody raised in a different species than the first one (recombinant rabbit monoclonal anti-p53, 1:100 dilution, DAKO/Agilent), was incubated overnight. Finally, a second Alexa Fluor 568-conjugated anti-rabbit secondary antibody was incubated for 1 h in the dark and slides were cover-slipped with an aqueous mounting media without DAPI (Vectashield Plus Antifade, Vector Laboratories), since both proteins were expected in the nucleus, and visualized in an Olympus FV100 confocal microscope.
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