Rabbit anti vwf

The collagen content of native valve leaflets was altered via targeted enzymatic digestion, similar to previous work described for hyaluronic acid depletion [5 (link)]. Leaflets were excised from porcine aortic valves (Hormel, Inc., Austin, MN) and washed in a solution of Medium 199 (pH 7.4) supplemented with 200 U/ml penicillin and 200 μg/ml streptomycin. This was followed by a 1.5 hour incubation at 37°C with shaking in a solution of 96 U/mL collagenase type II (Worthington Biochemical Corp.), while control leaflets were incubated in wash solution for the same duration of time. Leaflets were rinsed numerous times in wash solution, denuded of endothelial cells, cut in half, and prepared for either Day 0 characterization or in vitro organ culture as described in subsequent sections. Untreated native leaflets were used as controls. Endothelial denudation was confirmed via detection of von Willebrand factor (rabbit anti-vWF, Dako, Carpinteria, CA) using standard immunohistochemical techniques.

Rabbit anti-VWF was purchased from DAKO (cat. no. A0082; 1:10,000). Sheep anti-VWF (cat. no. AHP002; 1:10,000) and sheep anti-TGN46 (cat. no. AHP500; 1:200) were purchased from BIORAD. Sheep polyclonal anti-P-selectin was from R&D systems (cat. no. AF137; 1:100), mouse monoclonal anti-EEA1 (clone 14, cat. no. 610457; 1:200) and mouse anti-adaptin α (cat. no. 610502; 1:1000) was from BD Pharmingen, and mouse anti-clathrin light chain (CON.1, cat. no. AB9884; 1:100) and mouse anti-tubulin were from Sigma.

BOECs were seeded on 18 mm coverslips in 12-well plates (Sarstedt, Numbrecht, Germany). Cells were fixed with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA, USA) in phosphate-buffered saline (PBS) (Wisent Bioproducts, St. Bruno, QC, Canada). Immunofluorescence staining and imaging were done according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). Rabbit anti-VWF (DakoCytomation, Glostrup, Denmark) and goat anti-VECAD (Santa Cruz, Dallas, TX, USA) dilution 1:100 were used as primary antibodies. Corresponding species-specific Alexa Fluor 488- and Fluor 555-labeled antibodies (Thermo Fisher Scientific, Waltham, MA, USA) dilution 1:500 were used as secondary antibodies. Images were recorded by spinning disk confocal microscopy (Olympus IX81, Olympus corporation, Tokyo, Japan) under control of Volocity software (PerkinElmer, Groningen, The Netherlands).

hPIs in suspension and encapsulated in hydrogel maintained in culture for 1, 14 and 28 days were fixed in paraformaldehyde (PFA) 2% and 4% (w/v in PBS) and cryosectioned at 50 µm-thick via Cryostat (Histo-Line Laboratories). Sections were permeabilized with 0.3% Triton X-100 for 10 min at 4°C and blocked with 10% normal goat serum (NGS, Gibco) for 1 h at room temperature. The following primary antibodies were used: rabbit anti-insulin (1:300, ThermoFisher), mouse anti-chromogranin (1:100, ThermoFisher), mouse anti-glucagon (1:8000, Sigma-Aldrich), rabbit anti-Ki67 (1:750. Novus Biologicals), rabbit anti-vWF (1:500, DakoCytomation), mouse anti-fibroblast (1:200, Acris Antibodies), rabbit anti-collagen IV (1:100, Cedarlane), mouse anti-collagen I (1:2000, Sigma-Aldrich), rabbit anti-laminin (1:30, Sigma-Aldrich). To reveal primary antibodies, the following secondary antibodies were used: goat anti-rabbit Cy3 (1:1,000, Jackson), goat anti-mouse Cy3 (1:1,000, Jackson), goat anti-rabbit Alexa 488 (1:1,000, Invitrogen) and goat anti-mouse Alexa 488 (1:1,000, Invitrogen). Cell nuclei are stained with HOECHST 33342 (1:500, Molecular Probes).

Evoked WPB exocytosis was visualized microscopically by an anti-VWF antibody capture assay (Knop et al, 2004 (link)). Briefly, live cells grown at 37°C and 5% CO₂ were first incubated in rabbit anti-vWF (1:400; Dako)–containing medium for 20 min to saturate nonspecific antibody binding sites. Cells were then washed twice with warm PBS, new medium containing rabbit–anti–VWF-DyLight-650 conjugated antibodies was added and cells were incubated for 3 min. To induce VWF secretion, either histamine (100 μM) or ionomycin (10 μM) were added and incubation was continued for 15 min. The remaining steps were performed at RT and 3% BSA-containing PBS was used as buffer for blocking steps or to dilute antibodies. Cells were washed thrice, fixed in 4% PFA for 10 min, permeabilized in PBS containing 0.1% Triton X-100 for 2 min, washed thrice, and incubated with blocking buffer for 45 min. Mouse anti-VWF antibodies (clone F8/68, 1:400; Dako) were then added to label intracellular VWF and incubation was continued for 1 h at RT (or overnight at 4°C). Cells were then washed three times and incubated with goat antimouse AF514-conjugated antibodies (1:2,000) as secondary antibodies. Finally, cells were washed three times and mounted using mounting media.