After virus infection, cell monolayers were washed with BSS and fixed with 4% paraformaldehyde (pH 7.4) for 10 min at room temperature. Cells were then washed with 0.1 M phosphate buffer (pH 7.4), permeabilized with saponin (0.1 M phosphate buffer, 1% BSA, 0.6% saponin) for 10 min, washed again and incubated with the blocking buffer (0.1 M phosphate buffer, 1% BSA, 0.2% saponin) for 15 min. Next, cell monolayers were incubated for 1 h with 150 μL/well of specific antibodies, diluted with 0.1 M phosphate buffer with 0.1% BSA and 0.2% saponin, at 37 °C in a humid dark chamber. Detection of DENV2 antigens was performed using an anti-NS3 polyclonal serum, raised in rabbit inoculated with the recombinant DENV2 NS3 protein, or anti-envelope (E) antibodies obtained from rabbit immunized with a DNA vaccine encoding the ectodomain of the DENV2 E protein (pE1D2)63 (link). Subsequently, cells were washed with 0.1 M phosphate buffer and incubated for 1 h with anti-rabbit antibody (150 µL/well) conjugated with FITC (1:100 dilution, Southerm Biotechnology), at 37 °C in a humid dark chamber. Anti-GFAP antibody conjugated with TRITC fluorophore was also used for detection of astrocytes (1:200 dilution, Sigma). Slides were mounted with Vectashield medium (Vector Laboratories Inc., USA) and analyzed by fluorescence confocal microscope (Olympus FV10i-O).
Fv10i o
Manufactured by Olympus
Sourced in Japan
The FV10i-O is a confocal laser scanning microscope designed for live-cell imaging. It features a compact, integrated design and supports a range of objectives for various imaging applications.
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Lab products found in correlation
4 protocols using fv10i o
1
Immunofluorescence Staining for DENV2 Antigens
2
Immunofluorescence Staining of p53 and A11
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Cells (5 × 105 per chamber) were seeded into 12-chamber tissue culture slides. The next day, cells were rinsed with ice-cold PBS, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked in 5% BSA. Cells were subjected to immunofluorescence staining with either DO-7 or 1801 anti-p53 antibodies (1:400) and anti-A11 antibody (1:400) and incubated overnight at 4 °C. Cells were then incubated with goat anti-mouse IgG/Alexa 568–labeled anti-rabbit (1:800) or Alexa 488–labeled anti-mouse (1:800) secondary antibodies (Thermo Fisher Scientific) at room temperature for 1 h and finally stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were then examined using fluorescence confocal microscopy (FV10i-O, Olympus, Tokyo, Japan) and analyzed using FV10-ASW and ISI software.
3
Complement Activation in Liver Transplant
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Liver samples were obtained at the time of transplantation and stored in liquid nitrogen until immunofluorescence assays, sectioned at 5 μm, and stained with antibodies to membrane attack complex C5b/9 (clone: aE11, Santa Cruz cat #SC-58935, Brazil); the C3a component (clone: K13/16, Santa Cruz cat #SC-47688, Brazil) and C5a component (clone: 2952, Santa Cruz cat #SC-52634, Brazil) were applied to the sections at a dilution of 1 : 50 for 1 hour at 37°C. After incubation, sections were washed in phosphate-buffered saline (pH = 7.2) and incubated with the secondary antibody Alexia Fluor 488® (Abcam cat #AB-150113, USA) at a dilution of 1 : 1000. Photomicrographs were taken in a confocal microscope FV10i-O (Olympus, Japan) using FV10-ASW software. Calculation of marked areas was carried out using the software ImageJ (https://imagej.net/ImageJ ).
4
Uptake of Nanoparticles in Drug-Resistant Cells
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The uptake of nanoparticles in drug-resistant MCF-7/adr cells was detected by confocal laser microscopy and flow cytometry using coumarin as a fluorescence marker. In this section, nanoparticles were prepared using the same procedures by replacing paclitaxel with coumarin. Drug-resistant MCF-7/adr cells in the logarithmic growth phase were seeded into confocal dishes at a concentration of 1.5×105 cells/well and cultured for 24 h. Then the cells were treated with normal saline, coumarin nanoparticles, Ce6 nanoparticles, and dual sensitization anti-resistant nanoparticles at 2 μg/mL (in coumarin equivalence). After 6 h incubation, the medium in the confocal dishes was discarded, and the dishes were washed three times with PBS. The cells were counter-stained with Hoechst 33342 (2 μg/mL) for 10 min. The fluorescence intensity and distribution of coumarin in the cells were observed by a confocal microscope (FV 10i-O, Olympus, Tokyo, Japan).
MCF-7/adr cells were seeded at a density of 4×105 cells/well in 6-well culture plates. After 24 h incubation, the cells were exposed to normal saline, coumarin nanoparticles, Ce6 nanoparticles, and dual sensitization anti-resistant nanoparticles at 2 μg/mL in coumarin equivalence. After 1 h, 2 h, 4 h, and 8 h incubation, the cells were harvested from the plates, collected, and analyzed by a FACS Calibur™ flow cytometer (BD Biosciences, CA, USA).
MCF-7/adr cells were seeded at a density of 4×105 cells/well in 6-well culture plates. After 24 h incubation, the cells were exposed to normal saline, coumarin nanoparticles, Ce6 nanoparticles, and dual sensitization anti-resistant nanoparticles at 2 μg/mL in coumarin equivalence. After 1 h, 2 h, 4 h, and 8 h incubation, the cells were harvested from the plates, collected, and analyzed by a FACS Calibur™ flow cytometer (BD Biosciences, CA, USA).
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