Xenopus laevis embryos were obtained by standard in vitro fertilization protocols approved by Northwestern University Institutional Animal Care and Use Committee. Morpholinos were designed targeting Xenopus Deup1 and Cep63 (Gene Tools, LLC). The Deup1 morpholino had the sequence 5’-GGCTTTCAGTGTCTGTTTGCATTTC-3’ and the Cep63 morpholino had the sequence 5’-CATTCCGTTTTCTCAACACACTGCA-3’. Embryos were injected with 10–20 ng of morpholino and with Dextran-Cascade Blue (Thermo Fisher, D1976) as a tracer at the two to four-cell stage. Standard scrambled morpholino controls were used in all morpholino experiments (Gene Tools, LLC). For morpholino validation experiments, the morpholino-targetable Deup1-GFP or Cep63-GFP RNAs were injected 4 times in two-to four cell stage embryos, and then the corresponding morpholinos were injected as mosaics in two of the four blastomeres at the 4-cell stage with membrane RFP or dextran-blue as tracers.
Dextran cascade blue
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dextran-Cascade Blue is a fluorescent labeling agent used for the detection and visualization of biomolecules in various research and analytical applications. It is a water-soluble, high-molecular-weight dextran conjugated with the Cascade Blue fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Texas red dextran, by Thermo Fisher Scientific (3 mentions)
Sterile culture ware, by Thermo Fisher Scientific (3 mentions)
Cep63, by Gene Tools (2 mentions)
Microloader, by Eppendorf (2 mentions)
Phenol red free mem, by Thermo Fisher Scientific (2 mentions)
Anxv pacific blue, by Thermo Fisher Scientific (2 mentions)
Rat basophilic cells rbl, by American Type Culture Collection (2 mentions)
Glass micropipette, by Sutter Instruments (2 mentions)
Nih 3t3 fibroblasts, by American Type Culture Collection (2 mentions)
Fluorescein sodium salt, by Merck Group (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Wga alexa fluor 488 or 594 conjugate, by Thermo Fisher Scientific (2 mentions)
Cal520 dextran conjugate, by AAT Bioquest (2 mentions)
Vivo morpholino, by Gene Tools (2 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (2 mentions)
Rhodamine dextran, by Thermo Fisher Scientific (2 mentions)
μ slide 6 0.5 glass bottom, by Ibidi (1 mentions)
Fv1000mpe confocal imaging system, by Olympus (1 mentions)
Lysine fixable, by Thermo Fisher Scientific (1 mentions)
36 protocols using dextran cascade blue
1
Xenopus Embryo Morpholino Knockdown
2
Measuring Brain Edema and BBB Permeability
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Brain edema was measured with the wet‐dry method.15 Brains were quickly removed and weighed. Each brain was separated into ipsilateral and contralateral hemispheres and dried overnight at 95°C. Water content was calculated as % H2O=100×(wet‐dry weight)/wet weight. Brain edema was also visually determined by extravasation of the injected Evans blue dye. BBB permeability was assessed after 2 hours of femoral vein injection of fluorescein sodium salt (Sigma‐Aldrich), 5 kDa dextran–Cascade Blue, and 40 kDa dextran–fluorescein isothiocyanate (FITC) (Thermo‐Fisher Scientific, Waltham, MA) in mice in deep isoflurane anesthesia 22 hours after reperfusion. Before dextran injection, mice were injected with saline. The brain hemispheres were quickly separated to measure fluorescence after extraction by a methanol dye. To visualize leakage of the injected dye, brains were transcardially perfused with saline, fixed in 4% paraformaldehyde, embedded in Tissue‐Tek (OCT compound; Sakura, Horgen, Switzerland), and imaged with a confocal laser‐scanning microscope (LSM 510; Zeiss, Germany).
3
Lipid Membrane Probes and Cell Lines
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Synthetic lipids were obtained from Avanti. Di-4-ANEPPDHQ (Di4), BSA (fraction V), Phenol red-free MEM, AnxV-Pacific Blue, and dextran-cascade blue (3000 Da) were purchased from Thermo Fisher. NR12S was kindly provided by A. Klymchenko and Mikhail Bogdanov. All other chemicals were purchased from Sigma. Rat basophilic cells (RBL) and NIH 3T3 fibroblasts were purchased from ATCC. Glass micropipettes were from Sutter Instruments and microloaders from Eppendorf. For analysis of red blood cells, 6 channel IBIDI™ slides were used.
4
Fluorescent Dextran Uptake Assay
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Cells were seeded in ibi-treated μ-Slide VI 0.5 Glass Bottom (Ibidi) in complete media and incubated overnight. The following day, the cells were stimulated for 2 h with Dextran Cascade Blue 3 kDa, Dextran AF488 10 kDa, or Dextran tertamethylrhodamine 40 kDa (Thermo Fisher Scientific) at a concentration of 0.5 mg/mL. Then, the cells were washed twice with PBS and images were captured under a Zeiss LSM 780 confocal microscope.
5
Xenopus Embryo Morpholino Knockdown
6
Intravital Imaging of Tumors in Arg1-eYFP Mice
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We used the dorsal skinfold chamber to image tumor cells in Arg1-eYFP mice. For MC38-H2B-mApple and EL4-H2B-mApple tumors, 106 cells were injected in the fascia. Mice were monitored daily for 8 days prior to imaging. Where indicated, mice were injected i.v. with a Pacific Blue-labeled dextran nanoparticle (containing 1 nmol Pacific Blue dye) for TAM labeling or Pacific Blue-labeled 500 kDa amino-dextran (containing 56 nmol Pacific Blue dye) for vascular labeling 48 . Mice were anesthetized (2% isoflurane in oxygen) and immobilized on a custom heating platform to maintain body temperature and minimize motion artifacts. Imaging was performed with an Olympus FV1000MPE confocal imaging system and an XLUMPLFL 20x water immersion objective (NA 0.95; Olympus America). Pacific Blue, YFP and mApple were imaged sequentially using 405-nm, 473-nm and 559-nm light sources and BA430-455, BA490-540, and BA575-620 emission filters, along with DM405/473/559 nm dichroic beam splitters (Olympus America). Excised organs were also imaged on this microscope, but for these experiments Arg1-eYFP mice were injected 2.5 h prior with 75 μL of 10 kDa dextran-Cascade Blue (ThermoFisher), and 5 min prior with rhodamine-lectin (Vector Labs) for vascular labeling.
7
Lipid Membrane Probes and Cell Lines
8
Selective Depletion of Tissue Macrophages
9
Microfluidic Droplet Generation for Fluorescent Dye Monitoring
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To monitor the generation in real-time, the microfluidic chip was positionned on a brightfield microscope. The sample changer and the continuous phase were connected to the microfluidic inlets using PTFE tubing 1/16"x0.2 mm inner diameter (Cluzeau Info Labo, France). The continuous phase is composed of fluorinated oil (Novec-7500, 3M) containing 1% or 4 % (w/w) fluorosurfactant (Fluosurf, Emulseo, France) for isothermal amplification and PCR amplification, respectively. Samples were spiked with concentrations comprised between 0 and 200 nM of dextran-conjugated dye including Dextran Texas Red 70,000 MW, Dextran Alexa Fluor 488 3,000 MW, Dextran Alexa Fluor 647 10,000 MW, Dextran Cascade Blue 10,000 MW Lysine fixable (ThermoFisher Scientific). Droplets were generated by hydrodynamic flow focusing using a pressure pump controller (MFCS-EZ, Fluigent, France). Samples were emulsified serially by switching the tubing position using the external magnet to manipulate the sample rack. An air bubble and optionally a water slug (0.5 µL ~ 1.5 cm of PTFE tubing) were inserted between each sample. The droplets were collected at the outlet using a 200 µL pipet tip (LoRetention tip, eppendorf).
10
Fluorescent Tracers for Biomedical Imaging
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Sterile culture ware was obtained from Fisher Scientific (Pittsburgh, PA, USA), reagents and chemicals were purchased from Sigma-Aldrich or Bio-rad laboratories (Hercules, CA, USA), while Mini-Protean® TGX™ gels 4–15% (#456-1084) from Bio-rad laboratories was used for gel electrophoresis. Dextran-Cascade Blue® (10,000 MW; #D-1976) was obtained from Life Technologies, while Fluorescein isothiocyanate (FITC)-dextran (3,000–5,000 MW; #FD4) and Rhodamine B isothiocyanate (RITC)—dextran (70,000 MW; #R9379) were purchased from Sigma-Aldrich.
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