Pictures were acquired using a confocal microscope (Leica, FV3000 or LSM710). Each picture was taken using the scan format of 1024 × 1024 pixels, speed at 400 Hz, image x and y dimensions of 238.10 mm, voxel-size of 231.51 × 3 nm, average line 1, average frame 3, and pinhole 1. The lasers considered were of 405 nm, 488 nm, 594 nm, and 647 nm. Each picture was adjusted to a width of 6 cm and a resolution of 300 pixels/inch.
Fv3000
Manufactured by Leica
The FV3000 is a high-performance confocal microscope system designed for advanced imaging applications. It features a modular architecture, allowing for customization to meet specific research needs. The FV3000 delivers exceptional optical performance, enabling high-resolution, multi-dimensional imaging of a variety of sample types.
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Lab products found in correlation
Lsm710, by Leica (1 mentions)
Cm dii, by Thermo Fisher Scientific (1 mentions)
Sp dioc18, by Thermo Fisher Scientific (1 mentions)
Vt1000s vibratome, by Leica (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Sp8 system, by Leica (1 mentions)
Fv1000, by Leica (1 mentions)
Stellaris 8, by Leica (1 mentions)
Paraformaldehyde (pfa), by Absin (1 mentions)
Cell recovery solution, by Corning (1 mentions)
Ethanol, by Sangon (1 mentions)
5 protocols using fv3000
1
Confocal Microscopy Imaging Protocol
2
Two-color Somite Fate Mapping in Skate Embryos
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Two-color somite fate mapping experiments were performed as described in Criswell et al. (2017b) (link) and Ward et al. (2017) (link). S24 skate embryos were removed from their egg cases to a petri dish and anesthetized in tricaine (MS-222 1 mg/L in seawater). Adjacent somites were injected with the red-fluorescent lipophilic dye CM-DiI and the green-fluorescent lipophilic dye SpDiOC18 (ThermoFisher). Concentrated stocks of CM-DiI (5 µg/µL in absolute ethanol) and SpDiOC18 (2.23 µg/µL in dimethylformamide) were diluted 1:10 in 0.3 molar sucrose for injection. After injection embryos were returned to their egg cases and maintained in a flow-through seawater table at 15°C for 8–12 weeks post-injection.
Injected skate embryos were euthanized using an overdose of tricaine (1 g/L in seawater) and fixed in 4% paraformaldehyde overnight at 4°C. Embryos were then rinsed 3 × 5 min in phosphate-buffered saline (PBS), embedded in 15% gelatin in PBS and post-fixed in 4% paraformaldehyde in PBS for 4 nights at 4°C before vibratome sectioning. A Leica VT1000S vibratome was used to cut 100 µm sections of tissue in sagittal plane, which were then DAPI-stained (1 µg/mL), coverslipped with Fluoromount-G (Southern Biotech) and imaged on an Olympus FV3000 (trunk and transitional vertebrae) or Leica Sp5 (tail vertebrae) confocal microscope.
Injected skate embryos were euthanized using an overdose of tricaine (1 g/L in seawater) and fixed in 4% paraformaldehyde overnight at 4°C. Embryos were then rinsed 3 × 5 min in phosphate-buffered saline (PBS), embedded in 15% gelatin in PBS and post-fixed in 4% paraformaldehyde in PBS for 4 nights at 4°C before vibratome sectioning. A Leica VT1000S vibratome was used to cut 100 µm sections of tissue in sagittal plane, which were then DAPI-stained (1 µg/mL), coverslipped with Fluoromount-G (Southern Biotech) and imaged on an Olympus FV3000 (trunk and transitional vertebrae) or Leica Sp5 (tail vertebrae) confocal microscope.
3
Visualizing Plant Cell Walls and Nuclei
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Propidium iodide (PI) staining of leaf tissues was performed as described by Nguyen and McCurdy (2015) (link). For pAtSWEET11::AtSWEET11-GFP plants, cleared samples were stained with 0.05% (w/v) calcofluor white solution for 40 min. After staining, samples were washed in ClearSee solution for 30 min before being mounted on slides in ClearSee for confocal microscopy observation.
Three confocal microscopy systems were used for image collection in this study, namely the Olympus FV1000 and FV3000 systems and a Leica SP8 system. Imaging settings were as follows: PI, excitation: 552 nm, emission window: 570–670 nm; calcofluor white, excitation: 408 nm; emission window: 425–460 nm; GFP, excitation: 488 nm, emission window: 497–527 nm.
Three confocal microscopy systems were used for image collection in this study, namely the Olympus FV1000 and FV3000 systems and a Leica SP8 system. Imaging settings were as follows: PI, excitation: 552 nm, emission window: 570–670 nm; calcofluor white, excitation: 408 nm; emission window: 425–460 nm; GFP, excitation: 488 nm, emission window: 497–527 nm.
4
Whole-animal Fluorescent Imaging
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HCR in situ and antibody-stained samples were imaged using Olympus fv3000 and Leica Stellaris 8 microscopes using a ×40 objectives. Z-sections of whole animals were taken with the same laser power and settings for all HCR in situ animals.
5
Immunostaining of Liver Organoids
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Organoids were completely harvested using Cell Recovery Solution (Corning, USA). (1) IHC staining: organoids were washed once with PBS 1 ×, dehydrated in 70% ethanol (Sango Biotech, China), and stained in 0.5% eosin (Sango Biotech, China; dissolved in 96% ethanol) for 30 min. After rehydration in 100% ethanol, the organoids were embedded in paraffin. The subsequent procedures were the same as the normal IHC steps. The data were analyzed using ImageJ (https://imagej.net/Download ) (2) IF staining: after fixation with 4% (w/v) paraformaldehyde (Absin, China) at 4 °C, the organoids were washed in 0.1% (v/v) Tween-PBS (Sango Biotech, China) and blocked with TritonX-100-BSA solution. Primary antibodies were used to incubate the organoids in 24-well plates at 4 °C overnight. After one wash, the organoids were incubated with corresponding secondary antibodies, before finally being transferred to slides with coverslips and observed under laser scanning confocal fluorescence microscopy (LSCFM) (Leica, FV3000). The detailed procedures were described previously [63 (link),67 ].
Immunostaining of mouse liver tissue slides for markers, including cytokeratin 19 (CK19/Krt19), albumin (Alb), α-SMA, and IL-17A, was performed using a Novo-Light Multiplex Fluorescence Immunohistochemical Kit (WiSee Bio, China). The complete list of immunostaining antibodies used can be found in Table S1.
Immunostaining of mouse liver tissue slides for markers, including cytokeratin 19 (CK19/Krt19), albumin (Alb), α-SMA, and IL-17A, was performed using a Novo-Light Multiplex Fluorescence Immunohistochemical Kit (WiSee Bio, China). The complete list of immunostaining antibodies used can be found in Table S1.
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