Mice were subjected to deep anesthesia, and 200 μg of FITC‐conjugated isolectin B4 (Vector Laboratories Burlingame, CA, USA) were injected intracardially. The animals were then killed, and brain explants were established and incubated on cell culture inserts for at least 1 h. Alternatively, explants were incubated for 10 min with CD16/32 (1:300 dilution; BioLegend, San Diego, CA) to block IgG Fc receptors II/III, washed with PBS, and stained with phycoerythrin (PE)‐ or FITC‐conjugated antibodies to mouse CD31 (1:300, BioLegend) or to mouse Sca1 (1:300, BioLegend). Images were acquired with an FV10i confocal microscope (Olympus).
Fitc conjugated isolectin b4
Manufactured by Vector Laboratories
Sourced in United States, Germany
FITC-conjugated Isolectin B4 is a lectin derived from the seeds of the plant Griffonia simplicifolia. It is conjugated to the fluorescent dye FITC (Fluorescein Isothiocyanate). Isolectin B4 binds to alpha-D-galactose and N-acetyl-alpha-D-galactosamine residues, making it a useful tool for the detection and visualization of specific cell types and structures.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5, by Leica (2 mentions)
Ribogreen, by Thermo Fisher Scientific (2 mentions)
Fv10i confocal microscope, by Olympus (1 mentions)
Cd16 32, by BioLegend (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by GE Healthcare (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Triton x 100, by Vector Laboratories (1 mentions)
Ds fi1c camera, by Nikon (1 mentions)
Nis elements software, by Nikon (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay reagent, by Promega (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate dii, by Thermo Fisher Scientific (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindodicarbocyanine perchlorate did, by Thermo Fisher Scientific (1 mentions)
Hct116 human colorectal carcinoma cells, by American Type Culture Collection (1 mentions)
6 p toluidino 2 naphthalenesulfonic acid tns, by Fujifilm (1 mentions)
3 3 dioctadecyloxacarbocyanine perchlorate dio, by Thermo Fisher Scientific (1 mentions)
Cholesterol chol, by Merck Group (1 mentions)
9 protocols using fitc conjugated isolectin b4
1
Visualizing Brain Vasculature in Mice
2
Choroidal Angiogenesis Induction and Measurement
3
Visualizing Laser-Induced Choroidal Neovascularization
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After euthanasia on day 1, 3, 7, or 14 following laser photocoagulation, the eyes were enucleated and prefixed with 4% paraformaldehyde (Nacalai tesque, Kyoto, Japan) for 30 min. Retina-RPE-choroid complexes were microsurgically isolated from the prefixed eyes and further fixed with 4% paraformaldehyde for 1 h. The retina was removed from RPE-choroid complexes. The RPE-choroid complexes were then washed with PBS (1X; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4:Nacalai tesque) and incubated for 30 min with PBS blocking buffer containing 0.5% Triton X-100 (GE Healthcare UK, Buckinghamshire, UK) and 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO). To visualize neovascularization, RPE-choroid complexes were incubated overnight at 4 °C with 0.01% fluorescein isothiocyanate (FITC)-conjugated isolectin B4 derived from Griffonia (Bandeiraea) simplicifolia agglutinin (Vector Laboratories, Peterborough, UK) diluted with PBS containing 0.5% Triton X-100. After the RPE-choroid complexes were washed with PBS and sealed with VECTASHIELD (Vector Laboratories), CNV was observed with a fluorescence microscope (IX71; Olympus, Tokyo, Japan). Note that FITC-labeled isolectin binds to microglial cells and macrophages as well as endothelial vascular cells [19 (link)].
4
Retinal Vessel Visualization and Quantification
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To visualize retinal vessels, 6 retinas for each age group were incubated with FITC-conjugated isolectin B4 (1:200; Vector Laboratories, Burlingame, CA, USA) diluted in PBS containing 5% BSA and 2% Triton X-100. After 48 h incubation at 4 °C, retinas were rinsed in PBS and flat-mounted on polarized glass slides with the RGC layer facing up. Images of retinal vessels were obtained following scanning acquisition with a 10x Plan-Apochromat objective using an epifluorescence microscope (Ni-E; Nikon-Europe, Amstelveen, The Netherlands) equipped with a digital camera (DS-Fi1c camera, Nikon-Europe) and subsequent automatic digital reconstruction with NIS-Elements software (Nikon-Europe, Amstelveen, The Netherlands). Angio Tool software was used to quantify vessel density as percent of the area occupied by vessels and lacunarity as the size distribution of gaps or lacunae between vessels [28 (link)]. Values of vessel density and lacunarity were normalized to those measured at 2 months.
5
Oxygen-Induced Retinopathy and Nrp1 Deletion
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Litters were placed in 75% oxygen chamber fitted with an oxygen controller (PROOX 110; Reming Bioinstruments Co., Redfield, USA) from P7-12 and kept a room air until P17 [28 ]. Cre recombinase was activated by i.p. injection of 4-hydroxy-tamoxifen (OHT) at P11 (in the OIR model, 5μg/pup) or at P3 (2μg/pup to test Nrp1 deletion efficiency). Eyes were then processed for wholemount immunohistochemistry as previously described [30 (link)], using antibodies against Nrp1 (1:200, R&D systems, AF566), collagen IV (1:200, AbD Serotec, 2150–1470), conjugated α-smooth muscle actin (ASMA, 1:200, Cy3-conjugated, Sigma Aldrich, C6198) and FITC-conjugated isolectin B4 (1:200, Vector Labs). Wholemounts of retinas were imaged and analysed according to published protocols [32 ]. This involved manually tracing and quantifying the neovascular and avascular areas with ImageJ.
6
Quantifying Muscle Vascularization and ADSC Engraftment
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Tissue vascularization and ADSCs engraftment were evaluated at day 14 and 35. Mice were anesthetized and injected with 50 μg of FITC-conjugated isolectin B4 (FL-1201, Vector Laboratories) via tail vein. After 10 min, mice were perfused with PBS for 3 min to remove unbound isolectin B4 in the blood vessels, and perfusion-fixed with 4% paraformaldehyde. The gastrocnemius muscles were removed, fixed with 4% paraformaldehyde at 4°C overnight, and incubated in 30% sucrose solution for 24 h. Frozen sections of the tissue were obtained at 10 μm thickness and counterstained with DAPI. Capillary density and engraftment of injected ADSCs were analyzed by counting FITC and PKH positive cells, respectively, in five randomly selected fields per section. The experiments were performed in duplicate or triplicate for each human ADSCs sample.
7
Choroidal Angiogenesis Induction and Measurement
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Laser photocoagulation (OcuLight GL, IRIDEX, Mountain View, CA, USA) was performed on both eyes of mice to induce neovascularization, and on day 7 after injury, choroidal angiogenesis volumes were measured by scanning laser confocal microscopy (TCS SP5, Leica, Wetzlar, Germany) as previously reported with 0.7% FITC-conjugated Isolectin B4 (Vector Laboratories, Burlingame, CA, USA).24 (link),25 (link) For intravitreous administration in choroidal angiogenesis experiments, drugs was administered into the vitreous humor of mice using a 33-gauge double-calibre needle (Ito Corporation, Tokyo, Japan) as previously described.26 (link)
8
Fluorescent Probes for Cell Imaging
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EverFluor TMR-X was purchased from Setareh Biotech (Eugene, OR, USA).
6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS) was obtained from Wako Chemicals (Osaka, Japan). Ribogreen, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) were obtained from Molecular Probes (Eugene, OR, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). Dual-Luciferase Reporter Assay Reagent was purchased from Promega Corporation (Madison, WI, USA). FITC-conjugated Isolectin B4 was purchased from Vector Laboratories (Burlingame, CA). HeLa human cervical carcinoma cells was purchased from RIKEN Cell Bank (Tsukuba, Japan). HCT116 human colorectal carcinoma cells was purchased from ATCC (Manassas, VA, USA). All siRNAs ware obtained from Hokkaido System Science Co. Ltd. (Sapporo, Japan). The sequences for the sense and antisense strands of the siRNAs used in this study are listed in Supplementary Table.
6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS) was obtained from Wako Chemicals (Osaka, Japan). Ribogreen, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) were obtained from Molecular Probes (Eugene, OR, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). Dual-Luciferase Reporter Assay Reagent was purchased from Promega Corporation (Madison, WI, USA). FITC-conjugated Isolectin B4 was purchased from Vector Laboratories (Burlingame, CA). HeLa human cervical carcinoma cells was purchased from RIKEN Cell Bank (Tsukuba, Japan). HCT116 human colorectal carcinoma cells was purchased from ATCC (Manassas, VA, USA). All siRNAs ware obtained from Hokkaido System Science Co. Ltd. (Sapporo, Japan). The sequences for the sense and antisense strands of the siRNAs used in this study are listed in Supplementary Table.
9
Formulation and Characterization of pH-Sensitive Lipid Nanoparticles
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A pH-sensitive cationic lipid, YSK05, was synthesized as described previously [15] .
Cholesterol (chol) was purchased from SIGMA Aldrich (St. Louis, MO). 3,3′-Dioctadecyloxacarbocyanine perchlorate (DiO) and Ribogreen were purchased from Molecular Probes (Eugene, OR, USA). TNS was purchased from Wako Chemicals (Osaka, Japan). FITC-conjugated Isolectin B4 was purchased from Vector Laboratories (Burlingame, CA). The sequences for the sense and antisense strands of siRNAs used in this study are listed in Table S1.
Cholesterol (chol) was purchased from SIGMA Aldrich (St. Louis, MO). 3,3′-Dioctadecyloxacarbocyanine perchlorate (DiO) and Ribogreen were purchased from Molecular Probes (Eugene, OR, USA). TNS was purchased from Wako Chemicals (Osaka, Japan). FITC-conjugated Isolectin B4 was purchased from Vector Laboratories (Burlingame, CA). The sequences for the sense and antisense strands of siRNAs used in this study are listed in Table S1.
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