As our previous description [12 (link)], at P17, deeply anesthetized OIR mice were intracardially injected with a fluorescein-conjugated dextran (molecular weight = 500,000; Sigma-Aldrich) dissolved in PBS. After 1-hr perfusion, the eyes were enucleated and fixed in 4% paraformaldehyde for 1 hr. The retinas were dissected, flat-mounted and viewed by a fluorescein microscopy (BX50; Olympus, Tokyo, Japan) at a magnification of 40×.
Fluorescein conjugated dextran
Manufactured by Merck Group
Sourced in United States
Fluorescein-conjugated dextran is a fluorescent labeling agent used in various biological applications. It consists of the polysaccharide dextran conjugated with the fluorescent dye fluorescein. This product can be used to label and track molecules, cells, or other biological entities in research and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Mai tai hp, by Spectra-Physics (4 mentions)
Bz x710, by Keyence (3 mentions)
Movable objective microscope, by Olympus (3 mentions)
Xlumplanfl n, by Olympus (3 mentions)
Fluoview fv10i, by Olympus (2 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
Glass slide, by Matsunami (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Maitai hp laser, by Spectra-Physics (1 mentions)
Movable objective microscope, by Sutter Instruments (1 mentions)
Paraformaldehyde (pfa), by Biosesang (1 mentions)
Mscan software, by Sutter Instruments (1 mentions)
Facsverse, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
15 protocols using fluorescein conjugated dextran
1
Visualizing Retinal Vasculature in OIR Mice
2
Dendritic Cell Uptake of Dextran
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After treatment with LPS (100 ng/mL) and ALP (200 μg/mL) for 18 h, DCs were added to fluorescein-conjugated dextran (0.5 mg/mL, Sigma-Aldrich) for 30 min at 37 °C. The resulting cell pellets were then harvested and washed 2 times with cold FACS buffer (phosphate-buffered saline supplemented with 0.5% FBS and 0.1 sodium azide). Finally, the washed cells were labeled with DC surface antibody (anti-CD11c, PE-Cy7). The cells (Dextran+CD11c+ cells) were counted using FACSverse, and the FACS data were analyzed with the FlowJo V10 software.
3
Fluorescent Dextran Uptake Assay
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Bt WT and various mutants containing mCherry were cultured in BMM supplied with 2.5 mg/mL of D(+)-glucose and 2.5 mg/mL of dextran 35 kDa overnight anaerobically. The resulting cultures were then pelleted by centrifugation and washed twice with PBS. The pellets were then resuspended in 1× PBS, where the OD was measured. We then diluted the cells to a final OD of 0.1 in PBS and added fluorescein-conjugated dextran of various sizes (Sigma-Aldrich) to the solution at a final concentration of 1 mg/mL. We then incubated the solution aerobically for 1 hour, and the cells were then washed 3× in 1× PBS. Resulting cells were then resuspended in 1× PBS and imaged by fluorescence confocal microscopy. For each sample and replicate, five image frames across the agar pad were recorded. The relative abundance of mCherry- and sfGFP-expressing cells of signal corresponding to dextran binding and import was recorded for each single cell within each sample using the “analyze particle” function in Fiji. The ratio of sfGFP:mCherry was then quantified for every individual cell within an experiment and averaged per experiment.
4
Murine Choroidal Neovascularization Imaging
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After the FA, the mice were perfused with 0.5 mL PBS containing 20 mg/mL of fluorescein-conjugated dextran (MW = 2000 kDa, Sigma-Aldrich, St. Louis, MO, USA) for 5 min. The eyes were then removed and fixed with 4% paraformaldehyde (PFA) for 2 h at room temperature (RT). Then the lens, cornea, and retina were removed, and the RPE-choroid-sclera complex was flat-mounted on glass slide (Matsunami, Osaka, Japan) using Fluoromount (Diagnostic Bio Systems, Pleasanton, CA, USA). The CNV regions were examined and photographed with a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan). The size of the CNV lesions was measured in a masked manner.
5
Visualizing Retinal Vascular Network
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Deeply anesthetized OIR mice were intracardially injected with a fluorescein-conjugated dextran (molecular weight = 500,000; Sigma-Aldrich; St. Louis, MO, USA) in PBS. After 1 hour of perfusion, the eyes were enucleated and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 1 hour. The retinas were dissected, flat-mounted and viewed by fluorescence microscopy (Eclipse 80i; Nikon Instruments Inc. Tokyo, Japan) at 40× magnification.
6
Quantifying Choroidal Neovascularization in Mice
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After confirming that the mice were under deep anesthesia, mice were administrated 0.5 mL PBS containing 20 mg/mL fluorescein-conjugated dextran (MW ≈ 2000 kDa, Sigma-Aldrich) through the tail vein. Five minutes after administration of fluorescein-conjugated dextran, their eyes were enucleated and fixed in 4% paraformaldehyde for 12 h. The cornea and lens were removed while viewing the eye under a microscope, and the retinas were carefully peeled from the RPE-choroid-sclera complex. The RPE-choroid-sclera complexes were flat-mounted and covered with a micro cover glass (Matsunami Glass) after a few drops of fluoromount (DBS Diagnostic Biosystems) were placed on the microscope slide. The slides were photographed with BZ-X710 (Keyence) for the overall picture and FLUOVIEW FV10i (Olympus) for the laser spots. The areas of the CNV were measured using the ImageJ analysis software (National Institutes of Health).
7
Imaging Cerebral Vasculature in Mice
8
Imaging Cerebral Vasculature in Mice
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Mice were briefly anesthetized with isoflurane (5% in oxygen) and retro-orbitally injected with 50 μL 5% (weight/volume in saline) fluorescein-conjugated dextran (70 kDa, Sigma-Aldrich, cat.no.: 46945), and then fixed on a spherical treadmill. Imaging was done on a Sutter Movable Objective Microscope with a 20×, 1.0 NA water dipping objective (Olympus, XLUMPlanFLN). A MaiTai HP (Spectra-Physics, Santa Clara, CA) laser tuned to 800 nm was used for fluorophore excitation. All imaging with the water-immersion lens was done with room temperature distilled water between the PoRTS window and the objective. All the 2PLSM measurements were started at least 20 min after isoflurane exposure to reduce the disruption of physiological signals due to anesthetics. High-resolution image stacks of the vasculature were collected across a 500 by 500 μm field and up to a depth of 250 um from the pial surface. All the images were acquired with increasing laser power up to 100 mW at a depth of ~200 μm. Lateral sampling was 0.64 um per pixel and axial sampling was at 1 um steps between frames. Shortly (within 20 min) after the imaging, the mouse was perfused with FITC filling for STPT based ex vivo vasculature imaging.
9
Two-Photon Microscopy of Brain Vasculature
10
Quantifying Antigen Uptake in Dendritic Cells
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BMDCs were equilibrated at 37°C or 4°C for 45 min and then pulsed with fluorescein-conjugated dextran (40,000 Da) from Sigma-Aldrich (St. Louis, MO) at a concentration of 1 mg/ml. After several washes with cold phosphate-buffered solution, the cells were stained with a PE-Cy7-conjugated anti-CD11c mAb and then measured with a FACS LSRII to reveal antigen uptake. Nonspecific binding of dextran to DCs was determined by incubating DCs with FITC-conjugated dextran at 4°C, and the resulting background value was subtracted from the specific binding values.
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