Paclitaxel (Sigma-Aldrich, St Louis, MO, USA), was dissolved at 25 mg/mL in 1:1 Cremophor EL: ethanol and freshly diluted 1:12.5 in 0.9% sterile saline prior to injections. The retrograde tracer, DiI (Invitrogen, Carlsbad, CA, USA), was dissolved at 170 mg/mL in dimethylsufoxide (DMSO, Sigma-Aldrich) and diluted 1:10 in 0.9% sterile saline. FITC-conjugated Isolectin B4 (IB4, Sigma-Aldrich) was dissolved in dH20 as a stock solution of 1 mg/ml, and then diluted to a final concentration of 5 µg/ml in HEPES bath solution the day of use. Fura-2 acetoxymethyl (AM) ester (TEF Laboratories, Austin, TX, USA) was dissolved in DMSO as a 2.5 mM stock solution and diluted to a final concentration of 2.5 µM in HEPES bath solution. Pluronic F-127 (TEF Laboratories) was dissolved in DMSO as a 25% stock solution and diluted to 0.025% in HEPES bath solution. Capsaicin (Sigma-Aldrich) was dissolved in ethanol as a 10 mM stock solution and diluted to 500 nM in HEPES bath solution.
Fitc conjugated isolectin b4 ib4
Manufactured by Merck Group
FITC-conjugated Isolectin B4 (IB4) is a fluorescently-labeled lectin that binds to specific glycoconjugates on the cell surface. It is commonly used as a marker for endothelial cells and microglia in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Paclitaxel, by Merck Group (1 mentions)
Capsaicin, by Merck Group (1 mentions)
Antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
P mtor, by Abcam (1 mentions)
F4 80, by Abcam (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
3 protocols using fitc conjugated isolectin b4 ib4
1
Preparation and Application of Neurochemical Probes
2
Quantifying Choroidal Neovascularization in Mice
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After the mice were euthanized, their eyes were enucleated and then fixed with 4% PFA for 1 h at room temperature. The cornea and lens were discarded, and the neuroretina was carefully separated from the RCC via blunt dissection. The RCC was then permeabilized with 0.15% Triton X-100 in PBS for 20 min and blocked with 5% BSA in PBS for 1 h at room temperature. To label the CNV lesions, the RCC was incubated with FITC-conjugated isolectin-B4 (IB4, 10 μg/mL, Sigma–Aldrich) at 4 °C overnight. After washing with PBS, the whole RCC was flat-mounted with antifade mounting medium (Thermo Fisher Scientific). CNV lesions were photographed under a fluorescence microscope (Olympus) and the areas (μm2) of lesions were quantified using the ImageJ software in masked fashion. Lesions with obvious failure or hemorrhage were excluded for analysis35 . Immunofluorescence staining of F4/80 (1:100, Abcam) or p-mTOR (1:100, Abcam) was performed as that of IB4 except that fluorescent-labeled secondary antibodies were added.
3
Retinal Vascularization and Apoptosis in Newborns
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Newborn pups were administered with either AB‐Tie1‐39 or control IgG (40 mg/kg) intraperitoneally on postnatal days P2 and P4. On P6, eyeballs were fixed in methanol. Retinas were isolated and co‐stained with FITC‐conjugated isolectin B4 (IB‐4, Sigma, L9381) and cleaved caspase 3 (Cell Signaling, 9661). High‐resolution 3D images were acquired on a Zeiss LSM 710 confocal microscope. Quantitation of staining intensities was performed with Fiji Is Just ImageJ (FIJI) software. For analysis of the tip cells, the number of tip cells (identified by their filopodia) at the retinal front was manually counted and normalized to the radial length of the vascular front.
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