dextran was mixed at a 1:3 ratio of 70kDa Texas-red labeled dextran to 70 kDa dextran (ThermoFisher cat. D1830). The mixture of dextran was reacted with 2-ethoxypropene overnight. Mice were inoculated with 200,000 B16F10 tumor cells on day 0. On day 10, mice were treated with 10 μg of cGAMP encapsulated in Texas-red labeled Ace-DEX polymer. Texas-red Axe-DEX was made as described above with Texas-red labeled dextran obtained from Thermo Fisher (Waltham, MA cat. D1830). Twenty-four hours after treatment, mice were sacrificed the following organs were harvested: brain, lung, liver, kidney, spleen, lymph nodes, and tumor. The organs were then imaged using the IVIS Kinetic (PerkinElmer Waltham, MA). Samples were excited for 1 second at an excitation wavelength of 570 nm and detected using the Cy5.5 emission filter. The spleen was then taken and made into a single cell suspension. The splenocytes were stained (CD45 (BV-421), CD3 (PE-Cy7), CD4 (PerCP-cy5.5), CD8 (AF700), CD11b (APC), and CD11c (Pac-Blue); Biolegend, San Diego, CA), analyzed on a LSR II (BD Biosciences, San Jose, CA) and analyzed by FlowJo software (Tree Star, Ashland, OR).
Texas red labeled dextran
Manufactured by Thermo Fisher Scientific
Sourced in United States
Texas-red labeled dextran is a fluorescently labeled carbohydrate polymer used as a tool in biological research. It serves as a tracer and can be used to study fluid dynamics, cell permeability, and other processes within living systems.
Automatically generated - may contain errors
Lab products found in correlation
Dextran, by Thermo Fisher Scientific (2 mentions)
Zen acquisition software, by Zeiss (1 mentions)
Vt1200 vibratome, by Leica camera (1 mentions)
Fluorescein isothiocyanate fitc labeled dextran, by Merck Group (1 mentions)
Trimscope, by Miltenyi Biotec (1 mentions)
Matlab, by MathWorks (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Non specific mouse igg, by Jackson ImmunoResearch (1 mentions)
Iodogen pre coated tubes, by PerkinElmer (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Monoclonal mouse anti human icam 1 clone r6.5, by American Type Culture Collection (1 mentions)
Alginate hydrogel, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Epifluorescence microscope, by Nikon (1 mentions)
Lsrfortessatm cell analyzer, by BD (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Clariostar monochromator microplate reader, by BMG Labtech (1 mentions)
Lysotracker, by Zeiss (1 mentions)
Axioimager software, by Zeiss (1 mentions)
16 protocols using texas red labeled dextran
1
In Vivo Imaging of Dextran-Encapsulated cGAMP
2
Longitudinal Two-Photon Imaging of Mouse Visual Cortex
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Cranial window surgeries were performed as previously described within the visual cortex (2.5µm lateral and 2.0 µm posterior from bregma) (Goldey et al. 2014) . One week after surgery, mice were head-fixed to a custom-built running wheel and trained to run while head restrained for increasing time intervals several days a week. Two weeks post surgery long-term 2-photon live imaging began. Mice were given a retro-orbital injection of Texas Red labeled dextran (Fisher Scientific; Waltham, MA) 10 minutes prior to imaging and were head restrained on a custom built running wheel, which was positioned directly under the microscope objective.
Images were acquired with a 20X water immersion objective (Zeiss, NA 1.0) on a Zeiss Laser Scanning 7 MP microscope equipped with a tunable coherent Chameleon Ultra II multiphoton laser and BiG detector. Three different regions of interest (ROIs) were taken at least 75µm below the surface of the brain, with z-stacks spanning 45-65µm with a step size of 2.5µm for each animal. On the first day of imaging, each ROI was imaged every 5 minutes over 2 hours.
The same ROIs were then imaged once (single z-stack) on the following days post first imaging session: 1, 3, 7, 10, 14, 17, 21, 24, 28, 35, and 42 days. For each imaging day, the ROIs from day 0 of imaging were identified based on the vascular structure.
Images were acquired with a 20X water immersion objective (Zeiss, NA 1.0) on a Zeiss Laser Scanning 7 MP microscope equipped with a tunable coherent Chameleon Ultra II multiphoton laser and BiG detector. Three different regions of interest (ROIs) were taken at least 75µm below the surface of the brain, with z-stacks spanning 45-65µm with a step size of 2.5µm for each animal. On the first day of imaging, each ROI was imaged every 5 minutes over 2 hours.
The same ROIs were then imaged once (single z-stack) on the following days post first imaging session: 1, 3, 7, 10, 14, 17, 21, 24, 28, 35, and 42 days. For each imaging day, the ROIs from day 0 of imaging were identified based on the vascular structure.
3
Longitudinal Imaging of Brain Vasculature
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Mice were given a retro-orbital injection of Texas Red labeled dextran (Fisher Scientific; Waltham, MA) 10 minutes prior to sacrifice to label vasculature. Mice were euthanized at P7 or P≥120, brains were isolated and sectioned coronally at a thickness of 300µm using a Leica VT1200 vibratome in oxygenated 37°C artificial cerebrospinal fluid (ACSF). Slices were mounted on a MatTak glass bottom microwell dish and placed in a Zeiss Observer Spinning Disk Confocal microscope equipped with diode lasers (405nm, 488nm, 594nm, 647nm) and Zen acquisition software (Zeiss; Oberkochen, Germany). Image acquisition started after a minimum of 30 minutes of tissue equilibration at 37°C with 5% CO 2 and within 2 hours of decapitation.
Oxygenated ACSF was continuously perfused over the slices at a rate of 1.5-2µm/minute for the duration of equilibration and imaging. Per animal, one field of view was imaged every 5 minutes over 6 hours on an inverted Zeiss Observer Spinning Disk Confocal and a 20X objective. Zstacks spanning 50-60µm, with serial optical sections of 1.5-2µm were recorded from a minimal depth of 30µm beneath the surface of the slice to avoid cells activated by slicing.
Oxygenated ACSF was continuously perfused over the slices at a rate of 1.5-2µm/minute for the duration of equilibration and imaging. Per animal, one field of view was imaged every 5 minutes over 6 hours on an inverted Zeiss Observer Spinning Disk Confocal and a 20X objective. Zstacks spanning 50-60µm, with serial optical sections of 1.5-2µm were recorded from a minimal depth of 30µm beneath the surface of the slice to avoid cells activated by slicing.
4
Visualizing Cochlear Microcirculation with Multiphoton Microscopy
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Fluorescein isothiocyanate- (FITC-) labeled dextran (order number 46947; molecular weight 500 kDa; 0.05 to 0.1 mL of a 5% solution in 0.9% NaCl; Sigma, Deisenhofen, Germany) or Texas red-labeled dextran (order number D1830; molecular weight 70 kDa; 1.0 mL of a 5% solution in 0.9% NaCl; Life Technologies, Carlsbad, CA, USA) was injected intravenously as a plasma marker to visualize cochlear microcirculation. Multiphoton microscopy was performed on a TriMScope (LaVision BioTec, Bielefeld, Germany) described elsewhere [35 (link), 36 (link)].
Two water immersion objectives were used for image acquisition, either 20x (numerical aperture 0.95, working distance 2 mm, field number 22 mm, and field of view in current study 0.5 mm × 0.5 mm) or 10x magnification (numerical aperture 0.3, working distance 3.5 mm, field number 26.5 mm, and field of view in current study 1 mm × 1 mm). 0.9% NaCl or ultrasound gel was applied as immersion liquid. Excitation was achieved with 800, 860, or 1180 nm.
Two water immersion objectives were used for image acquisition, either 20x (numerical aperture 0.95, working distance 2 mm, field number 22 mm, and field of view in current study 0.5 mm × 0.5 mm) or 10x magnification (numerical aperture 0.3, working distance 3.5 mm, field number 26.5 mm, and field of view in current study 1 mm × 1 mm). 0.9% NaCl or ultrasound gel was applied as immersion liquid. Excitation was achieved with 800, 860, or 1180 nm.
5
Intestinal Organoid Permeability Assay
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Intestinal organoids were exposed to mTNFα for 24 h and then incubated in 1 mg/ml Texas Red labeled dextran (average weight 10 kDa; Life Technologies) for 30 min. Following incubation, excess dye was removed by serial washes with PBS and the plate imaged immediately. Wells were scored for fraction of dye-retaining intestinal organoids. 30–50 intestinal organoids were seeded per well. Eight wells per experiment were scored for each condition. Opened intestinal organoids were excluded from analysis. Results were validated by a blinded, independent observer.
6
Intranasal Dextran Delivery in Mice
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In the control group (group 1), no dextran was administered.
In the IN sham group (group 2) and IN treatment group (group 3), ∼2 mg of 40 kDa Texas Red-labeled dextran (Life Technologies Inc, Grand Island, NY, USA) was administered intranasally following procedures used before [4] (link), [30] (link). The dextran was dissolved in saline at a concentration of 40 mg/mL. The anaesthetized mice were placed supine with the head position stabilized horizontally. A micropipette was used to intranasally administer 3 µL drops of the dextran solution to alternating nostril every 2 minutes. Drops were placed at the opening of the nostril, allowing the animal to snort each drop into the nasal cavity. A total of 51 µL dextran solution (∼2 mg dextran) was delivered over a course of 34 min.
For the IV treatment group (group 4), the same amount of dextran (51 µL in volume, 40 mg/mL in concentration, and ∼2 mg in dose) was administered by IV bolus injection through the tail vein.
In the IN sham group (group 2) and IN treatment group (group 3), ∼2 mg of 40 kDa Texas Red-labeled dextran (Life Technologies Inc, Grand Island, NY, USA) was administered intranasally following procedures used before [4] (link), [30] (link). The dextran was dissolved in saline at a concentration of 40 mg/mL. The anaesthetized mice were placed supine with the head position stabilized horizontally. A micropipette was used to intranasally administer 3 µL drops of the dextran solution to alternating nostril every 2 minutes. Drops were placed at the opening of the nostril, allowing the animal to snort each drop into the nasal cavity. A total of 51 µL dextran solution (∼2 mg dextran) was delivered over a course of 34 min.
For the IV treatment group (group 4), the same amount of dextran (51 µL in volume, 40 mg/mL in concentration, and ∼2 mg in dose) was administered by IV bolus injection through the tail vein.
7
Brain Barrier Disruption Quantification
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To detect blood–brain barrier disruption at various timepoints, mice receiving five repetitive CBIs (“repCBI-hf”) were sacrificed either one, three, or seven days after the last trauma compared to a group of mice receiving sham surgeries. Under low-light conditions, mice were perfused with Texas-red labeled dextran (40 kDa, D1829, Invitrogen, Waltham, MA, USA) solved in saline containing heparin [63 (link)]. Brains were extracted and shock-frozen in methylbuthane. Using a cryostat, 30 µm thick coronal slices across the hippocampus were cut. In addition to counterstaining with Hoechst as described above, capillaries were stained using Lectin (Lycopersicon Esculentum (Tomato) Lectin, DyLight 488, 1:400, Invitrogen L32470). Sections were imaged using a confocal microscope (TCS SP8, Leica, Wetzlar, Germany).
A region of interest (ROI) outlining the hippocampus was manually drawn. Using a custom script written using MATLAB (2020b, MathWorks, Massachusetts), a mask was generated based on the Lectin-positive capillaries (Figure S1G ) to mask out intravascular dextran signal (Figure S1H ). The remaining images were used to compute histograms of intensity values across the red color channel (RGB brightness intensities between 0 and 255) and its mean intensity values were compared across groups.
A region of interest (ROI) outlining the hippocampus was manually drawn. Using a custom script written using MATLAB (2020b, MathWorks, Massachusetts), a mask was generated based on the Lectin-positive capillaries (
8
Quantifying ICAM-1 Expression in Endothelial Cells
9
Spatial Bioprinting of Fluorescent Hydrogels
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To demonstrate the spatial position capabilities of our DVDOD bioprinter, we used two dispensing units loaded with bioink composed of low viscosity (≤12 cP) alginate hydrogel (Sigma-Aldrich, St. Louis, MO, USA) mixed with FITC and Texas Red labeled dextran (Invitrogen, Carlsbad, CA, USA), respectively. Different patterns of droplets were used. To prevent liquid drying during the assay, 1 µL droplet was used to dispense onto the surface of a Petri dish. Images were captured using an epifluorescence microscope (Nikon, Minato City, Tokyo, Japan).
10
Quantifying Vascular Leakage in Mice
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Vascular leakage was analyzed as previously described (21 (link), 23 (link)). β1- or α4-integrin antibodies, or control antibodies (all 2.5 mg⋅kg−1), were administered to 2- to 4-mo-old male C57BL/6 mice by i.p. injection. Cre-positive tamoxifen-induced Itgb1WT/fl (Itgb1WT/iΔEC) mice were compared with Cre-negative Itgb1WT/WT, Itgb1WT/fl, or Itgb1fl/fl littermates or Cre-positive Itgb1WT/WT (control mice). To induce endotoxemia, LPS (O55:B5, 11 mg⋅kg−1) was administered i.p. for 16 h. Alexa Fluor-594–labeled fluorospheres (100 nm) or Texas red-labeled dextran (70 kDa; Invitrogen, ThermoFisher Scientific) was injected via the tail vein and allowed to circulate for 4 min, followed by sequential perfusion with PBS and 1% paraformahdehyde (PFA) in PBS via the left ventricle. Tracheas and ears were immunostained, and the fluorescence was quantified as described in SI Appendix, SI Materials and Methods .
Fluorescence microscopy and quantification of fluorescent and TEM images are described inSI Appendix, SI Materials and Methods .
Fluorescence microscopy and quantification of fluorescent and TEM images are described in
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