The cells were fixed in 4% (m:v) paraformaldehyde for 15 min at 4°C, washed with PBS, and then permeabilized with 0.3% Triton X-100 for 10 min at room temperature (RT). After non-specific binding was blocked with 1% BSA, the cells were incubated with primary antibody at 4°C in a humid chamber overnight. The cells were then incubated with a secondary antibody conjugated to FITC or TRITC for 1h at RT. After the cells were washed with PBS, images were taken with an Olympus inverted fluorescent microscope with CCD camera.
Inverted fluorescent microscope
Manufactured by Olympus
Sourced in Japan, China
The Inverted Fluorescent Microscope is a laboratory instrument designed for observing fluorescently labeled samples. It features an inverted configuration, where the light source and objectives are positioned below the specimen stage, allowing for easy access and manipulation of the sample during observation.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Axiovert 100 microscope, by Zeiss (2 mentions)
Tcs spe confocal laser scanning microscope, by Leica (2 mentions)
Dsu spinning disk confocal scanner, by Olympus (2 mentions)
Phalloidin tritc, by Merck Group (2 mentions)
Matrigel, by Corning (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
5 ethynyl 2 deoxyuridine (edu), by RiboBio (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Cy3 labeled secondary antibody, by Beyotime (2 mentions)
Alexa fluor 488 conjugated goat anti human igg antibody, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Solarbio (2 mentions)
Ros assay kit, by Beyotime (2 mentions)
Mouse and rabbit isotype controls, by Thermo Fisher Scientific (1 mentions)
Flow cytometry, by BD (1 mentions)
Thioflavin s, by Yuanye Bio-Technology (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Hoechst 33342, by Olympus (1 mentions)
Lentivirus, by Hanbio Biotechnology (1 mentions)
113 protocols using inverted fluorescent microscope
1
Immunocytochemistry of Fixed Cells
2
Microscopy Imaging and Processing Protocol
3
Immunofluorescence Analysis of Stem Cell Cultures
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Undifferentiated iPSCs cultures, trilineage differentiation cultures and cardiomyocyte cultures were fixed with 4% paraformaldehyde for 15 min and permeabilized by incubating in DPBS with 1% BSA and 0.1% Triton X-100 for 15 min at room temperature. iPSCs were labeled for pluripotent stem cell marker OCT3/4. Trilineage differentiation cultures were evaluated for markers of ectoderm (Pax6), mesoderm (Brachyury) and endoderm (AFP). Cardiomyocytes were evaluated for markers (cTnT and α-actinin). Samples were incubated in primary antibodies at 4 °C overnight, followed by incubation in secondary antibodies at room temperature for 60 min. Mouse and rabbit isotype controls (ThermoFisher Scientific, Waltham, MA, USA) were used in the control experiments. Nuclei were stained with Hoechst stain. Samples were studied using inverted fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan).
4
Osteogenic Differentiation Immunofluorescence Protocol
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The transfected cells were inoculated into a 12-well plate with a coverslip in each hole and cultured in mineralized medium for 7 days. After washing with PBS twice, the cells were fixed with 4% paraformaldehyde for 30 min. The cells were then penetrated with 0.25% Triton-100 at room temperature for 12 min, washed several times with PBS, and sealed with goat serum (DCS/BioGenex, Hamburg, Germany) at 37°C for 45 min. The cells were then incubated with an antibody against RUNX2 and ALP at 4°C overnight, and then incubated with a fluorescence-labeled secondary antibody at room temperature for 45 min, and the nuclei were dyed with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) for 90 s. Images were observed using an inverted fluorescent microscope (Olympus, Shanghai, China).
5
Mitochondrial Membrane Potential Assay
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MMP was assessed using JC-1 dye. In normal cells, the dye aggregates upon polarization membrane showing orange-red fluorescent. If the MMP dissipates, the dye cannot enter into the transmembrane space, remaining its monomeric form of green. Briefly, the trypsinized cells were centrifuged at 400 g for 5 min, washed with phosphate-buffered saline (PBS), and then incubated with 0.5 mL JC-1 working solution per tube at 37 °C for 30 min. Fluorescent microscopic images of PC12 cells were obtained under an inverted fluorescent microscope (Olympus, Tokyo, Japan) with × 40 objective. In addition, the intensities of red and green fluorescence were also determined by flow cytometry (BD Biosciences, San Jose, USA) at an excitation/emission value of 490/525 nm. The data were expressed as a red/green fluorescence ratio (set to 100% in control).
6
Quantifying Vascular Protein Expression
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To estimate the expression of GLUT 1 and eNOS proteins in the blood vessels, immunofluorescence and immunohistochemical staining were used, with reference to the literature.26 (link) Images were randomly captured by using an inverted fluorescent microscope (Olympus, Japan) with a charge coupled device camera system at fixed exposure times for comparison. The statistical analyses of incorporation of GLUT 1 and eNOS in thoracic aortas were calculated using ImagePro Plus software (Bethesda, MD, USA).
7
Thioflavin S Staining of Amyloid Deposits
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Thioflavin S staining can detect amyloid deposits, and the operation is as described before [38 (link)]. After the mice were sacrificed, brain tissue was separated, and half of the brain was cut and placed in a vial filled with paraformaldehyde (4% (v/v), pH 7.4) for 24 h. Then, it was embedded in paraffin and cut into 5 μm slices. The xylene, 100%, 90%, 80%, and 70% ethanol were used for washing for 5 min, followed by 5 min × 3 times PBS (pH 7.4) washing. The sections were permeated in permeate for 15 min and boiled for 20 min, followed by 5 min × 3 times PBS washing. The 0.5% Thioflavin S (CAS#1326-12-1; Shanghai Yuanye Bio-Technology Co., Ltd) was added to the sections for 8 min. After that, the sections were rinsed with 5 min × 3 times PBS, 50% ethanol, and PBS again, respectively. Finally, the sections were mounted with DAPI anti-fluorescence. The sections were observed, and images were acquired by an inverted fluorescent microscope (Olympus, Tokyo, Japan). Image J analysis software (National Institutes of Health, Scion Corporation, USA) was used for measuring the amyloid plaque area ratio in the cortex and CA1 regions of the hippocampus.
8
Cell Proliferation Assay Protocol
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Cell proliferation was detected using the Cell-Light™ EdU Cell Proliferation Assay Kit (Ruibo Biotechnology, Guangzhou, China) according to the manufacturer’s instructions. U251 and GBM1 cells were treated with vehicle or with different concentrations (10 μM and 20 μM) of SU4312 for 24 h. The cells were incubated with 50 μM EdU for 2 h, fixed in 4% paraformaldehyde for 15 min, and then treated with 0.5% Triton X-100 for 20 min. Thereafter, cells were incubated in 1 × Apollo® reaction mixture for 30 min, followed by DAPI staining for 15 min. After being washed with phosphate buffered saline (PBS) three times, the cells were observed with an inverted fluorescent microscope (Olympus, Tokyo, Japan) and photographed.
9
Structural Analysis of Pretreated KR
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To understand the structural changes occurred before and after pretreatment, auto-fluorescence imaging of KR was performed using inverted-fluorescent microscope (Olympus, Japan). The intensity of the red and green fluorescence was calculated using image J software.
10
Endothelial Cell Tubulogenesis Assay
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Matrigel with reduced growth factors was pipetted into pre-chilled 96-well plate (50 μl Matrigel per well) and polymerized for 30 min at 37°C. PAECs following different treatments (2 × 104 cells per well) were resuspended in 100 μl of basic media, and seeded in Matrigel-coated 96-well plate. After 4–6 h of incubation, tubular structures were photographed using Olympus inverted fluorescent microscope with a 20× magnification. The number of branch points was quantified in technical triplicate.
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