Zebrafish larvae were collected at 4 or 5 dpf and fixed in 4% paraformaldehyde. Lipophilic dyes, DiD, and DiI (D7757 and D282 from Thermo Fisher), were separately micro-injected into the retinal ganglion cell layer of the two eyes. The dyes were allowed to diffuse along retinal axons overnight at 4°C or 1–2 h at 37°C. The fluorescently labeled retinal projections were acquired on a confocal laser scanning microscope (Olympus FV1000). The images were presented as maximum projection of z-stacks.
D7757
Manufactured by Thermo Fisher Scientific
The D7757 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for essential laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information from Thermo Fisher Scientific would be required to describe the core function of this product accurately without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000, by Olympus (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
D12731, by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Cg15xh1, by Thorlabs (1 mentions)
Attofluor cell chamber, by Thermo Fisher Scientific (1 mentions)
8 protocols using d7757
1
Retinal Axon Tracing in Zebrafish
2
Paramecium Staining and Quantification
3
Microscopic Analysis of Liposome Lipofectamine and Cell Morphology
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For bright field microscopy of liposome, lipofectamine 2000 (Invitrogen) was diluted into Opti-MEM (Invitrogen) for 30 min at RT to allow formation of liposome. The mix was then transferred into DMEM in 24-well plates with or without FAC. The samples were incubated for 1 h at 37 °C and stained with DiD (50 μM, D7757, Thermo Fisher Scientific) before bright field microscope (Olympus IX73) observation.
For bright field cell morphology observation, A549 or Hela cells were seeded into 24-well plates and treated with Dynasore and/or FAC for indicated time (50 μM and/or 100 μM for 3 h, 80 μM and/or 100 μM for 6 h or 12 h) in DMEM without FBS. Cells were then fixed by 4% PFA, stained with 0.1% crystal violet and analyzed by Olympus IX73 microscope.
All image data shown were representative of at least five randomly selected fields from at least three independent experiments.
For bright field cell morphology observation, A549 or Hela cells were seeded into 24-well plates and treated with Dynasore and/or FAC for indicated time (50 μM and/or 100 μM for 3 h, 80 μM and/or 100 μM for 6 h or 12 h) in DMEM without FBS. Cells were then fixed by 4% PFA, stained with 0.1% crystal violet and analyzed by Olympus IX73 microscope.
All image data shown were representative of at least five randomly selected fields from at least three independent experiments.
4
Fluorescent Labeling of Extracellular Vesicles
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Freshly isolated EVs were incubated with either 1 μM of the fluorescent lipophilic tracer DiR (D12731, LifeTechnologies) or 1 μM of DiD (D7757, LifeTechnologies) at 4 °C for 30 min. 100 μL of each sample were placed on top of a size exclusion column (Exo-spin®) and centrifuged at 50×g for 1 min. 200 µL of PBS were then placed on top of the column and the EVs were obtained after centrifugation at 50×g for 1 min.
5
Fluorescent Mg-based Micromotors Synthesis
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For performing the characterization of the Mg-based micromotors along with the in vivo retention studies, fluorescent Mg-based micromotors were prepared by combining both 1% PLGA and 0.05% Chit solutions with 5 µg mL−1 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD, λex = 644 nm/λem = 665 nm, Life Technologies, D7757) and 1 µg mL−1 fluorescein isothiocyanate-dextran (FITC, λex = 492 nm/λem = 520 nm, Sigma-Aldrich, 46945) dyes, respectively. To compare with the Mg-based micromotors, inert silica (Si) microparticles (Nanocs, Inc., Cat. No. Si01-20u-1; 20 µm size) were used as core particles, following the same protocol described above.
6
TIRF Microscopy Calibration Protocol
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On every experimental day, two types of correction files were generated. (1) Cairn image splitter calibration—stack of 10 images of the NanoGrid (Miraloma Tech, LLC, A00020) with transmitted light illumination. This is required for image splitting and registration. (2) Flat field correction—To correct for the inhomogeneity of the TIRF excitation field, a stack of 10 images of fluorescein (Excited by 488 nm laser—ACROS ORGANICS, 2321-07-5) and DiD (Excited by 647 nm laser—Invitrogen, D7757) were acquired. Stock fluorescein was prepared in 1 M NaOH at 1 mg/ml and diluted at 5 μl/ml in NaOH on the day of imaging. DiD was resuspended according to manufacture instructions and diluted at 5 μl/ml in EtOH. The dilutions were applied on clean 25 mm #1.5H glass coverslips (Thorlabs, CG15XH1—coverslips were scratched in middle using a blade to find the focal plane that matches plasma membrane-coverslip interface) and mounted into Attofluor Cell Chambers (Invitrogen, A7816). TIRF images of both coverslips were taken separately with simultaneous 488 and 647 nm excitation to mimic live-cell imaging conditions.
7
Nanoparticle-Mediated Cell Targeting
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Targeting efficacy was evaluated using DiD (Invitrogen, catalog # D7757)-loaded fluorescent nanoparticles (DiD-NPs) with surface-conjugated antibody fragments. Whole splenocyte cultures were incubated for 30 mins with DiD-loaded, antibody fragment decorated nanoparticles suspended in PBS via flow cytometry (250,000 cells per 7.45 × 107 ± 9.45 × 106 NPs/mL) unless otherwise indicated.
8
Embryonic Somite Cell Labeling
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Eggs containing embryos at HH‐stages 17–18 were windowed and injected with India ink (Rotring, diluted 1:10 with 1xPBS buffer) into the yolk underneath the embryo for visualization. Depending on the stage of the embryo, a series of 5–8 somites were injected in the somitocoel or underneath the dermomyotome with a cell‐labelling solution (DID´ in DMF [2 mM], Invitrogen D7757) using injection needles made from borosilicate glass capillaries (O.D. 1.5 mm; I.D. 1.10 mm; Science Products GmbH) drawn on a Sutter P‐97 Puller. Thus, both sclerotome and dermomyotome/myotome cells were labelled.
The injected embryos were covered with 1–1,5 ml 1xPBS, the egg window closed with adhesive tape and reincubated until they had reached the desired stage. Embryos at HH‐stages 24–30 were isolated, prepared as described above and fixed with 4% paraformaldehyde. Specimens were examined and photographed using a Leica Thunder stereomicroscope. Where applicable, after the cell labelling procedure embryos were additionally subject to immunohistochemistry as described above.
The injected embryos were covered with 1–1,5 ml 1xPBS, the egg window closed with adhesive tape and reincubated until they had reached the desired stage. Embryos at HH‐stages 24–30 were isolated, prepared as described above and fixed with 4% paraformaldehyde. Specimens were examined and photographed using a Leica Thunder stereomicroscope. Where applicable, after the cell labelling procedure embryos were additionally subject to immunohistochemistry as described above.
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