Fitc conjugated isolectin b4

Sections (50 μm-thick) were rinsed in Tris-HCl phosphate buffer and then co-incubated overnight at room temperature with lectin-diluted with 1% BSA in Tris-HCl phosphate buffer or with the following primary antibodies: anti-vimentin (1:200, Millipore, Darmstadt, Germany); anti-βIII-tubulin (1:1000, Promega) and FITC-conjugated isolectin B4 (1:10, Sigma, St. Louis, MO, USA). After the sections were rinsed, they were co-incubated for 2 h at room temperature with the following fluorophore-conjugated secondary antibodies: Cy5-conjugated chicken anti-IgG (1:200, Jackson ImmunoResearch, Baltimore Pike, West Grove, PA, USA) and DyLight 547-conjugated mouse anti-IgG (1:200, Jackson ImmunoResearch). The sections were also incubated in Hoechst 33258 (Sigma, St. Louis, MO, USA) as a nuclear stain. Multi-labeled images were obtained by confocal spectral microscopy (LSM 780, Carl Zeiss). Z-sectioning was performed at 1 μm intervals, and optical stacks of at least 30 images were used for the analysis. Digital three-dimensional (3D) reconstructions were created using Zeiss LSM software (ZEN, Carl Zeiss Microscopy GmbH, Aalen, Germany).

Mouse retinas from post-natal day 5 (p5) eyes were dissected and flat-mounted on Millicell Cell Culture Inserts (Millipore) as described previously (Sawamiphak et al. 2010 (link)). Briefly, retinas were cultured in DMEM with 10% FCS at 35 °C in a humidified incubator (5% CO₂) until attached to the membrane (2–4 h). XAV939 was diluted in 3% FCS DMEM to a final concentration of 5 μM. Retinal explants were then subjected to XAV939 treatment (16 h) at 35 °C in a humidified incubator (5% CO₂). Retinas were fixed in 4% PFA at 35 °C for 4 h, blocked with 3% bovine serum albumin (BSA), and permeabilized with 0.1% Triton X-100 (both in CytoTBS-T buffer (20 mM Tris hydrochloride (Tris-HCl), 154 mM sodium chloride (NaCl), 2 mM ethylene glycol tetraacetic acid (EGTA), 2 mM magnesium chloride (MgCl₂), 0.1% v/v Tween 20, pH 7.2)) and stained with FITC-conjugated isolectin-B4 (1:200, L9381, Sigma-Aldrich) overnight at 4 °C. To visualize the retinal vasculature, images were acquired with a microscope (Zeiss, LSM700). Animal care and experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement and were approved by the local government (I-18/21 (“Interne Anzeige,” Regierungspräsidium Karlsruhe, Germany). All efforts were made to reduce the animal number as well as the animal suffering.

EPCs with inhibition or overexpression of Ifi204 were cultured in methylcellulose-based medium (MethoCultM3236; Stem Cell Technologies, Vancouver, BC, Canada) supplemented with VEGF (50 ng/ml; PeproTech, Rocky Hill, NJ, USA), SCF (100 ng/ml; PeproTech), IL-3 (20 ng/ml; PeproTech), EGF (50 ng/ml; PeproTech), bFGF (50 ng/ml; PeproTech), IGF-1 (50 ng/ml; PeproTech), and 30 % FBS (Sigma). EPC colonies were manually counted under a phase contrast microscope (Olympus) after culturing 1000 cells for 14 days. Endothelial phenotype of the EPC colonies was confirmed by uptake of DiI-Ac-LDL and FITC-conjugated isolectin B4 (Sigma Chemical Co., Milwaukee, WI, USA).

Three weeks after cell transplantation, the mice were sacrificed and the adductor muscles were harvested from the ischemic limbs. Samples were embedded in OCT compound, snap-frozen in liquid nitrogen, and cut into 10 µm-thick sections. Three sections from each mouse were stained with FITC-conjugated isolectin B4 (Sigma) and DAPI (Invitrogen), observed under a confocal microscope (Leica, Wetzlar, Germany). Four fields from each tissue section were randomly selected, and the number of capillaries was counted. Co-localization of CM-DiI-labeled cells with isolectin B4-labeled endothelial cells was also observed. Three representative fields were recorded and the percentages of donor-derived endothelial, non-endothelial cells and that of recipient-derived endothelial cells were calculated by Image-Pro Plus software (Media Cybernetics).

EPC colony formation was assessed by culturing 500 KSL cells in triplicate in methylcellulose-containing medium M3236 (Stem Cell Technologies) supplemented with; 100 ng/ml SCF (PeproTech), 50 ng/ml VEGF (PeproTech), 20 ng/ml interleukin-3 (R&D Systems), 50 ng/ml basic fibroblast growth factor (bFGF, R&D Systems), 50 ng/ml epidermal growth factor (EGF, PeproTech), 2 U/ml heparin (Sigma Aldrich), 30% FBS, and antibiotics. Small- and large-colonies (CFUs) after culture for 12 days were defined as focused clusters of rounded cells or as cell clusters with a central core of round cells and elongated sprouting cells at their peripheries [51 (link)]. Endothelial characteristics of the attached small- and large-CFUs were examined after uptake of DiI-conjugated Ac-LDL (DiI-Ac-LDL) (Biomedical Technologies Inc., MA) and binding with FITC-conjugated isolectin B4 (Sigma Chemical Co., WI), a standard marker of endothelial lineage cells. In our previous study, we defined these CFUs for the expression of additional marker genes such as CD31, Flk-1, eNOS [7 (link)], and in this study von Wilibrand factor (vWF), and VE-cadherin.

Pups were killed at P4 or P6, eyeballs were isolated and fixed in either methanol at −20 °C overnight or in 4% or 2% paraformaldehyde (PFA)/PBS for 1 h at RT. Isolated retinas were blocked and permeabilized with retina blocking buffer (10% normal goat serum, Dako, #X0907, in 0.5% Triton-X 100 and 1% BSA in PBS) for 1 h at RT. The retinal vasculature was stained with Alexa488- or FITC-conjugated Isolectin B4 (Sigma, 1:100) and NG2 antibody (Millipore, #Ab5320, 1:1,000) overnight at 4 °C. Subsequently, retinas were stained with the appropriate fluorescently labelled secondary antibodies and flat-mounted on microscope slides. Images were taken with the confocal microscopes Zeiss LSM710 and image analysis was accomplished with Fiji (ImageJ). The relative vessel area was calculated as Isolectin B4-positive area per retina area. Junctions and branches were counted in eight individual 600 μm × 600 μm fields of comparable regions in the retina and normalized to the vessel area. Pericyte coverage was determined by measuring the NG2-positive area associated with the vasculature and correlating it to the vessel area. If depicted as relative value, the single values per mouse were normalized to the average of the littermate WT or control treated animals. Each litter was analysed separately.