Stained coverslips were mounted on glass slides in ProLong Gold Antifade Reagent and examined using Fluoview FV10i laser scanning confocal microscope (Olympus Poland Sp. z o.o.) with 60× water-immersion objective, using ultraviolet/visible light LD lasers. Images were captured using FV10i software (resolution 1024 × 1024) and converted to 24-bit tiff files for visualization. Images were collected using z-stack imaging and analyzed using FV10i software (Olympus), the Fiji version of the free image processing software ImageJ (NIH Image, version 1.53a, Bethesda, MD, USA), and Adobe Photoshop CS6 software (Adobe Systems Incorporated, ver. 13.0, San Jose, CA, USA).
Fluoview fv10i confocal laser scanning microscope
Manufactured by Olympus
Sourced in Japan, United States
The FluoView FV10i is a confocal laser scanning microscope manufactured by Olympus. It is designed for high-resolution imaging of fluorescently labeled samples. The microscope utilizes laser excitation and a pinhole aperture to achieve optical sectioning, allowing for the capture of detailed, three-dimensional images.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Fv10i software, by Olympus (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Dapi stain, by Merck Group (2 mentions)
Rhodamine redtm x, by Jackson ImmunoResearch (2 mentions)
Bisbenzimide hoechst 33342, by Merck Group (1 mentions)
Lab tek 2, by Thermo Fisher Scientific (1 mentions)
Permanox lab tek chamber slide, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Fluoromount plus, by Cosmo Bio (1 mentions)
Alexafluor 488 conjugated goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Ba f8, by Developmental Studies Hybridoma Bank (1 mentions)
Sc 71, by Developmental Studies Hybridoma Bank (1 mentions)
Acti stain 488 phalloidin, by Cytoskeleton (1 mentions)
Mab4470, by R&D Systems (1 mentions)
Svec4 10, by American Type Culture Collection (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Alexa fluor 594 labeled donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
38 protocols using fluoview fv10i confocal laser scanning microscope
1
Confocal Microscopy Image Acquisition
2
Quantifying Aptamer-DOX Cellular Uptake
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Twenty-four hours before labeling, MDA-MB-231 and HEK293T cells were seeded at a density of 7.5 × 104 cells/cm2 in an eight-chamber slide (LabTekII; Nunc). Following removal of media, cells were incubated in binding buffer at 37°C for 30 min. Cells were then incubated with 1 μM DOX or aptamer-DOX conjugates at an equivalent DOX concentration of 1 μM for 60 and 120 min at 37°C. Bisbenzimide Hoechst 33342 (3 μg/mL; Sigma) was added to the cells during the final 10 min of incubation. Following each time point, the aptamer-DOX and DOX solutions were removed and cells were washed three times for 5 min each in binding buffer before visualization using a FluoView FV10i laser scanning confocal microscope (Olympus). To establish aptamer-DOX retention, following 120 min of incubation, cells were incubated for a further 24 h in DMEM before reimaging. The captured images were then analyzed using ImageJ to quantify DOX fluorescence. Area, integrated density, and mean gray value were measured for cells of interest (15 cells) and background. Corrected total cell fluorescence (CTCF) was calculated using the following formula: CTCF = integrated density − (area of selected cell × mean fluorescence of background readings).
3
Microscopic Analysis of Macrophage Mitochondria
4
Muscle Fiber Typing and Quantification
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Frozen sections of the soleus muscle (10 µm) were blocked with 5% normal goat serum in phosphate-buffered saline containing 0.1% Triton X-100 for 30 min at room temperature and incubated for 120 min at room temperature with anti-MHC I (1:300, dilution, BA-F8, Developmental Studies Hybridoma Bank, Iowa, USA) and anti-MHC IIa (1:300 dilution, SC-71, Developmental Studies Hybridoma Bank) antibodies. Sections were washed three times with phosphate-buffered saline for 5 min each and then incubated with Alexa Fluor 555-conjugated goat anti-mouse Ig2b (1:1,000 dilution, Invitrogen, Carlsbad, CA) for MHC I or Alexa Fluor 488-conjugated goat anti-mouse IgG1 (1:1,000 dilution, Invitrogen) for MHC IIa for 60 min at room temperature. Sections were washed and covered with Fluoromount/Plus (COSMO BIO CO., LTD., Tokyo, Japan). The fluorescence images were detected and captured with a FLUOVIEW FV10i laser scanning confocal microscope (OLYMPUS, Tokyo, Japan). The number of muscle fibers and cross-sectional areas were determined in 3 randomly selected square fields (840 × 840 µm) from each section using WinROOF image processing software (Mitani Corp., Tokyo, Japan).
5
Immunocytochemistry of Differentiated Muscle Cells
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After differentiation induction, cells grown on coverslips in a 24-well plate were fixed
with 2% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) for 30 min, washed with PBS, and
permeabilized with 0.1% Triton-X-100 (Nacalai Tesque) for 15 min. After washing with PBS,
the cells were blocked with 2% bovine serum albumin (BSA) for 15 min to eliminate
nonspecific reactions. An anti-mouse MyHC monoclonal antibody (MAB4470; R&D Systems,
Minneapolis, MN, USA) diluted at a ratio of 1:50 (final concentration 10 µg/ml) in PBS was
used as the primary antibody. Cells were incubated with the primary antibody for 1 hr at
room temperature. After washing with PBS, the cells were incubated with the secondary
CFTM-568-conjugated goat anti-mouse IgG antibody (Biotium, Hayward, CA, USA)
diluted in PBS at a ratio of 1:200 for 15 min. Acti-stainTM 488 Phalloidin
(Cytoskeleton, Denver, CO, USA) was added to the secondary antibody reaction solution for
F-actin staining. After washing with PBS, nuclear staining was performed using 5 µg/ml
Hoechst 33342 (Dojindo, Kumamoto, Japan) for 15 min, and the slides were then mounted with
Fluoromount/Plus™ (Diagnostic BioSystems, Pleasanton, CA, USA). Cells were analyzed using
a Fluoview FV10i laser scanning confocal microscope (Olympus, Tokyo, Japan).
with 2% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) for 30 min, washed with PBS, and
permeabilized with 0.1% Triton-X-100 (Nacalai Tesque) for 15 min. After washing with PBS,
the cells were blocked with 2% bovine serum albumin (BSA) for 15 min to eliminate
nonspecific reactions. An anti-mouse MyHC monoclonal antibody (MAB4470; R&D Systems,
Minneapolis, MN, USA) diluted at a ratio of 1:50 (final concentration 10 µg/ml) in PBS was
used as the primary antibody. Cells were incubated with the primary antibody for 1 hr at
room temperature. After washing with PBS, the cells were incubated with the secondary
CFTM-568-conjugated goat anti-mouse IgG antibody (Biotium, Hayward, CA, USA)
diluted in PBS at a ratio of 1:200 for 15 min. Acti-stainTM 488 Phalloidin
(Cytoskeleton, Denver, CO, USA) was added to the secondary antibody reaction solution for
F-actin staining. After washing with PBS, nuclear staining was performed using 5 µg/ml
Hoechst 33342 (Dojindo, Kumamoto, Japan) for 15 min, and the slides were then mounted with
Fluoromount/Plus™ (Diagnostic BioSystems, Pleasanton, CA, USA). Cells were analyzed using
a Fluoview FV10i laser scanning confocal microscope (Olympus, Tokyo, Japan).
6
Visualizing Mouse Endothelial Cell Cytoskeleton
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Mouse endothelial cells, SVEC4-10 (ATCC CRL-2181), 1×104 cells per well, were inoculated in the chamber slide. The cultured cells were treated with 2DG for 24 h. After treatment, the samples were washed with PBS and fixed in 3.7% formaldehyde for 15 min, followed by an additional wash with PBS for 2 min three times, and then permeabilized with 0.1% Triton X-100 in PBS for 10 min. After permeabilization, the cell samples were blocked with 1% bovine serum albumin, followed by washing with PBS. The slides were then incubated with CellMask and fluorescein phalloidin (fluorescent phallotoxins, 1:100; Molecular Probes, Thermo Fisher Scientific Inc.) for 20 min, followed by washing with PBS and mounted in DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Photomicrographs were taken using a FluoView FV10i laser scanning confocal microscope (Olympus, Tokyo, Japan).
7
Immunofluorescence Imaging of EV71 Infection
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Cells were seeded on glass coverslips, and were infected with EV71 at the indicated time, followed by fixation with 4% paraformaldehyde for 15 min at room temperature. After being washed twice with PBS, the cells were treated with specific primary antibodies against the proteins. The respective secondary antibody was Alexa Fluor 594‐labeled donkey anti‐mouse IgG (Molecular Probes, Life technologies) diluted at 1 : 1000. Images were acquired with an Olympus Fluoview FV10i laser scanning confocal microscope (Tokyo, Japan).
8
Synthesis and Characterization of Organic Compounds
9
Confocal Microscopy Imaging Protocol
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An LSM‐880 laser‐scanning confocal microscope (Zeiss, Jena, Germany) and Fluoview‐FV10i laser‐scanning confocal microscope (Olympus, Tokyo, Japan) were used. GFP or monomeric RFP fluorescence was excited by the 488 nm argon laser or the 561 nm He–Ne laser, respectively. Settings of the confocal microscope were the same for all image capture processes.
10
Visualizing Astrocyte Endocytic Pathways
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C8-D1A cells were seeded on 22-mm glass coverslips in 24-well plates. Astrocytes were left untreated or treated with poly(I:C) for indicated time points, fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.5% Triton X-100 (Merck KGaA) in PBS for 15 min. The cells were blocked with 3% bovine serum albumin (BSA, Merck KGaA) with 0.1% Triton X-100 in PBS. Next, astrocytes were stained with antibodies against EEA1, Rab7, LAMP1 or TLR3 for 1 h and subsequently incubated with FITC- and rhodamine Red-X-conjugated secondary antibodies for 1 h in the dark. The nuclei were stained with 1 μg/ml Hoechst 33342 (Merck KGaA) for 5 min in the dark. Lastly, slides were mounted with ProLong Gold Antifade Reagent (Thermo Fisher Scientific). The cells were observed using Fluoview FV10i laser scanning confocal microscope (Olympus) under a 60× water-immersion objective. Images were captured using FV10i software (Olympus), converted to 24-bit tiff files for visualization and analyzed with ImageJ (NIH Image, version 1.50i) and/or QuickPHOTO MICRO (Promicra, Czech Republic, version 3.1) software. To confirm the results obtained in fluorescent confocal microscopy, analogous experiments were performed using confocal microscopy-independent technology, Duolink®in situ proximity ligation assay PLA (Merck KGaA).
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