Using T3 stable lines of PHT1;1-GFP transgenic plants and their variants, GFP fluorescence was observed with confocal microscopy (Olympus IX83-FV3000) The seeds were sown in a nutrient medium for 4 d and then transferred to 1/2 MS with or without 100 µ M Na 2 HAsO 4 for 24 h. Seedlings were stained for 10 s with 10 μ M propidium iodide (PI) before imaging. Three lines were selected for each transgenic plant, and fluorescence was observed using a confocal microscope (Olympus IX83-FV3000). EGFP fluorescence was visualized by scanning with a 488 nm laser at 750 [V] PMT voltage and 500 to 540 nm spectral detection. And mCherry fluorescence for PI dye was visualized by scanning with a 561 nm laser at 655 [V] PMT voltage and 570 to 670 nm spectral detection. The relative signal content ratio of each fluorescence is obtained by ImageJ. Three independent replicates were selected for statistical analysis. The relative intensities of fluorescence were presented as the means.
Ix83 fv3000
Manufactured by Olympus
Sourced in Japan
The IX83-FV3000 is a high-performance inverted fluorescence microscope designed for advanced imaging applications. It features a modular and flexible design, allowing for customization to meet the specific needs of various research and laboratory settings.
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Lab products found in correlation
Mmp13, by Abcam (2 mentions)
Xds 1b, by Olympus (1 mentions)
Cm1860, by Leica camera (1 mentions)
Ab78547, by Abcam (1 mentions)
Ab34712, by Abcam (1 mentions)
Ab27156, by Abcam (1 mentions)
Vs200, by Olympus (1 mentions)
Ab955, by Abcam (1 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
Tissue tek cryo oct, by Thermo Fisher Scientific (1 mentions)
Smz18, by Nikon (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Trim16, by Santa Cruz Biotechnology (1 mentions)
Click it rna alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Forma co2 incubator, by Thermo Fisher Scientific (1 mentions)
Gsdmd, by Abcam (1 mentions)
Gsdme, by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
37 protocols using ix83 fv3000
1
Visualizing Arsenic Stress Response in PHT1;1-GFP Plants
2
Skeletal Muscle Organoid Imaging
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The images of skeletal muscle organoid were taken by inverted microscope (OLYMPUS XDS-1B) or a stereo microscope (OLYMPUS XTZ-D). Fluorescent images were captured using Olympus FV3000 Laser scanning confocal microscope (OLYMPUS IX83-FV3000). Transmitted electron microscopy images were taken by Cryo-electron microscope (Spirit 120 kV).
3
In Vivo Intestinal Uptake of Nanoparticles
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Male Sprague–Dawley (SD) rats (6–8 weeks, 230–250 g) were fasted overnight but had free access to water. Then, they were anesthetized with chloral hydrate, and the abdominal cavity was opened along the medioventral line. Two-centimeter sections of small intestinal loops were ligated at both ends. Then, 100 μL of the 1 mg/mL FITC-labeled PNC NPs and Chs-PNC NPs were administered into different intestinal loops of the same SD rats (n = 3). A quantity of 100 μL of PBS without NPs was used as the control group (pH = 7.4). After 2 h, the SD rats were euthanatized, and the loops treated with the FITC-labeled NPs were withdrawn and washed with PBS. After fixation in 4% paraformaldehyde for 4 h and dehydration in 30% sucrose overnight, the loops were embedded in an optimal cutting temperature compound and sectioned into 10 μm slices using a freezing microtome (Leica CM1860, Wetzlar, Germany). The cell nuclei were stained with Hoechst 33342 for 10 min and visualized using CLSM (Olympus, IX83-FV3000, Japan).
4
Bimolecular Fluorescence Complementation Assay
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The BiFC assay was conducted as previously described75 (link). Briefly, Agrobacterium was resuspended in a buffer (10 mM MES, 10 mM MgCl2, pH = 5.6) and mixed to a specific concentration. The YFPn-CIPK1 and PYR1/PYLs-YFPc were transformed into N. benthamiana leaves and expressed for 4 d. Fluorescence signals were captured using a confocal laser scanning microscope (Olympus IX83-FV3000). Tobacco protoplasts BiFC assay was conducted as previously described47 (link).
5
Hypoxia-Induced Cell Response
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1.5 × 105 cells were seeded onto 3.5 cm Petri dishes and cultured in RPMI 1640 medium with 10% (v/v) FBS and incubated for 12 h. The tested compounds were diluted to 5 μM, then the cells were incubated for 1 h under an atmosphere of 5% CO2 in air, thereafter the cells were incubated for different hours under an atmosphere of 5% CO2 and 1% O2. The medium was removed and washed with fresh medium. Confocal Fluorescence images were then obtained on Olympus IX83-FV3000.
6
Cellular Response to Hypoxia Exposure
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1.5 × 105 cells were seeded onto 3.5 cm Petri dishes and cultured in corresponding medium with 10% (v/v) FBS and incubated for 12 h. The tested compounds were diluted to 5 μM, then the cells were incubated for 1 h under an atmosphere of 5% CO2 in air, thereafter the cells were incubated for 6 h under an atmosphere of 5% CO2 and 1% O2 (hypoxia) or for 6 h under an atmosphere of 5% CO2 in air (normoxia). The medium was removed and washed with fresh medium. Confocal Fluorescence images were then obtained on Olympus IX83-FV3000.
7
Transient Tobacco Leaf Transformation
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The coding sequences of RchUGT169 and VENUS (with YFP marker) were recombined into the KpnⅠ and BamHⅠ sites in the pFUERTE vector [36 (link)], pFUERTE-RchUGT169-VENUS constructs were transformed into Agrobacterium tumefaciens (AGL1 + pSoup). RchUGT169 yellow fluorescent protein (YFP) was used for the transient transformation of tobacco leaves, and the pFUERTE-VENUS vector was used as the control. A laser scanning confocal microscope (IX83-FV3000, OLYMPUS, Tokyo, Japan) was used to observe the instantaneous transformation of tobacco leaves. OLYMPUS FV31S-SW v2.6 software was used to record images. The argon ion laser lines used were 488 nm for YFP and chlorophyll, and the fluorescence of YFP and chlorophyll was detected at 495–530 nm and 650–680 nm, respectively.
8
Transformation of Wheat Mesophyll Cells
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The transformation and expression of mesophyll cell protoplasts were based on the published method (Yoo et al. 2007 (link)). In brief, Mesophyll cell protoplasts were prepared by leaf digestion of wild-type plant Fielder Triticum aestivum seedlings under dark conditions. TaNRAMP3-GFP fusion constructs were transformed into Triticum aestivum mesophyll cell protoplasts by polyethylene glycol induction. The transformed protoplasts were incubated in darkness at 23 °C for 12–15 h, and then 60x objective lens was used (Olympus IX83-FV3000; Japan). The excitation wavelength was 488 nm, and the emission wavelength was 500–530 nm. The ratio of PM to intracellular signal was analyzed by ImageJ.
9
Immunohistochemistry and Immunofluorescence Staining
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IHC staining and IF staining were performed using standard procedures [39 (link)]. For immunostaining, slices were covered in 0.01 M sodium citrate buffer at 65 °C for 10 h for antigen recovery, subjected to 0.5% Triton-X 100 for 20 min to break the cell membranes, and then incubated with 10% FBS for 1 h to block non-specific binding sites. Incubation with primary antibody was performed according to the instructions, and the used primary antibodies included pTBK1 (5483, CST), CD68 (ab955, Abcam), Tommo20 (ab78547, Abcam), dsDNA (ab27156, Abcam), and Col2 (ab34712, Abcam).
For IHC staining, sections were subjected to corresponding horseradish peroxidase (HRP)-labeled secondary antibodies for 1 h. Diaminobenzidine (DAB) solution was utilized to visualize the target protein. The images were then captured with a microscope (VS200, Olympus, Japan). For IF staining, slices were incubated with fluorescent secondary antibody at 37 °C for 1 h. The slices were then stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed with a confocal fluorescence microscope (IX83-FV3000, Olympus, Japan). Quantitative analysis was performed with ImageJ. The proportion of positively stained cells was counted and averaged on at least 3 sections for analysis. Approximately 20 immunopositively stained cells were counted on each section.
For IHC staining, sections were subjected to corresponding horseradish peroxidase (HRP)-labeled secondary antibodies for 1 h. Diaminobenzidine (DAB) solution was utilized to visualize the target protein. The images were then captured with a microscope (VS200, Olympus, Japan). For IF staining, slices were incubated with fluorescent secondary antibody at 37 °C for 1 h. The slices were then stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed with a confocal fluorescence microscope (IX83-FV3000, Olympus, Japan). Quantitative analysis was performed with ImageJ. The proportion of positively stained cells was counted and averaged on at least 3 sections for analysis. Approximately 20 immunopositively stained cells were counted on each section.
10
Cytotoxicity and Biocompatibility Evaluation of Polydopamine Nanoparticles
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A live/dead cell viability assay was performed after the incubation of RAW264.7 cells in 1 mg/mL PDA NPs for 24 h to evaluate the cytotoxicity of PDA NPs. Then, the proportion of live cells was calculated from images obtained with a confocal fluorescence microscope (IX83-FV3000, Olympus, Japan). Besides, the Cell Counting Kit-8 (CCK8, Beyotime) was used according to the instructions. RAW264.7 cells were treated with 1 mg/mL PDA NPs for 24 h and then cultured in CCK8 staining solution for 2 h. The optical density at 450 nm was measured to determine the relative viable cell numbers.
C57/B6 mice (male, 12-week old) were used to evaluate the biocompatibility of NPs in vivo. Intra-articular injection of the following drugs was performed for the knee joints at a dose of 5 μL once per week: 1 mg/mL PEI-PDA NPs, 1 mg/mL PDA@C-176 NPs, and 1 mg/mL PEI-PDA@C-176 NPs. Untreated mice were used as sham controls. The mice were weighed every 6 days and sacrificed 30 days after the first injection. Blood erythrocytes, leukocytes, and hemoglobin were measured after the mice were sacrificed. The livers, kidneys, lungs, spleens, and hearts of sacrificed mice were harvested and embedded for histological analysis.
C57/B6 mice (male, 12-week old) were used to evaluate the biocompatibility of NPs in vivo. Intra-articular injection of the following drugs was performed for the knee joints at a dose of 5 μL once per week: 1 mg/mL PEI-PDA NPs, 1 mg/mL PDA@C-176 NPs, and 1 mg/mL PEI-PDA@C-176 NPs. Untreated mice were used as sham controls. The mice were weighed every 6 days and sacrificed 30 days after the first injection. Blood erythrocytes, leukocytes, and hemoglobin were measured after the mice were sacrificed. The livers, kidneys, lungs, spleens, and hearts of sacrificed mice were harvested and embedded for histological analysis.
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