Dylight 649 labelled ulex europaeus agglutinin 1 uea

Samples were fixed with 4% paraformaldehyde and permeabilized with a PBS solution containing Triton X-100 (5% v/v), sucrose (10% w/v), and magnesium chloride (0.6% w/v) for 1 hour each at room temperature. AlexaFluor 488 phalloidin (Life Technologies, Carlsbad, CA) was utilized to visualize F-actin. 4’, 6-diamidino-2-phenylindole (DAPI, 1 μg ml⁻¹) was utilized to visualize cell nucleus. For proliferation studies, EdU was applied for the final 24 hours prior to fixation for each study. EdU fluorescent labelling was performed following the manufacturer’s protocol (ClickIT EdU, Life Technologies). DyLight 649 labelled Ulex Europaeus Agglutinin-1 (UEA, 1:200, Vector Labs, Burlingame, CA) was utilized to visualize endothelial cell morphology in samples stained with EdU due to EdU ClickIT incompatibility with phalloidin staining. To visualize VE-cadherin, samples were sequentially blocked in bovine serum albumin (0.3% w/v), incubated with primary mouse monoclonal anti-VE-cadherin (200 ng ml⁻¹, Santa Cruz Biotechnology), and incubated with secondary AlexaFluor 647 goat anti-mouse IgG (H+L) (2 μg ml⁻¹, Life Technologies) each for 1 hour at room temperature. Fluorescently labelled collagen hydrogels were prepared as in Doyle⁶⁵ .

Samples were fixed with 4% paraformaldehyde and permeabilized with a PBS solution containing Triton X-100 (5% v/v), sucrose (10% w/v), and magnesium chloride (0.6% w/v) for 1 hour each at room temperature. AlexaFluor 488 phalloidin (Life Technologies, Carlsbad, CA) was utilized to visualize F-actin. 4’, 6-diamidino-2-phenylindole (DAPI, 1 μg ml⁻¹) was utilized to visualize cell nucleus. For proliferation studies, EdU was applied for the final 24 hours prior to fixation for each study. EdU fluorescent labelling was performed following the manufacturer’s protocol (ClickIT EdU, Life Technologies). DyLight 649 labelled Ulex Europaeus Agglutinin-1 (UEA, 1:200, Vector Labs, Burlingame, CA) was utilized to visualize endothelial cell morphology in samples stained with EdU due to EdU ClickIT incompatibility with phalloidin staining. To visualize VE-cadherin, samples were sequentially blocked in bovine serum albumin (0.3% w/v), incubated with primary mouse monoclonal anti-VE-cadherin (1:1000, Santa Cruz Biotechnology), and incubated with secondary AlexaFluor 647 goat anti-mouse IgG (H+L) (1:1000, Life Technologies) each for 1 hour at room temperature. To visualize mouse endothelial cells in explant hydrogels, samples were stained with Griffonia Simplicifolia Lectin I isolectin B4, DyLight 649 (1:200, Vector Labs).