Rabbit anti human vwf

Whole constructs were fixated in paraformaldehyde (4%), for 20 min, and then permeabilized with 0.3% Triton X-100 (Bio Lab Ltd.), for 10 min. Constructs were then washed with PBS and immersed overnight in BSA solution (5%; Millipore). Samples were then incubated with goat antihuman VE-cadherin (1:100; Santa Cruz), mouse antihuman Yes-associated protein (YAP) (1:100; Santa Cruz), mouse antihuman NG2 (1:100; Santa Cruz), rabbit antihuman vWF (1:150; Abcam), rabbit antihuman β-catenin (1:100; Sigma-Aldrich), or mouse antihuman Ki67 (1:20, DAKO) antibodies, overnight, at 4°C. Constructs were then treated with Cy3-labeled (1:100; Jackson Immunoresearch Laboratory), Cy5-labeled (1:100; Jackson Immunoresearch Laboratory), or Alexa-488 (1:400; ThermoFisher Scientific) antibodies, mixed with DAPI (1:1000; Sigma-Aldrich), for 2 h, at room temperature. For the phalloidin staining, constructs were treated with FITC phalloidin (1:100; Sigma-Aldrich) and DAPI for 20 min. For the mitomycin experiment, mitomycin-treated cells and control cells without mitomycin were fixated in paraformaldehyde (4%), for 20 min on day 10 of culture, and then incubated in a 30% (wt/vol) sucrose solution overnight, embedded in optimal cutting temperature compound (Tissue-Tek) and frozen for subsequent cryosectioning (5–20 µm). Standard protocols were used for H&E and Masson’s trichrome staining of the sections.

Immediately after supernatant removal, cells were washed 3X with PBS (ThermoFisher Scientific, Wilmington, DE, United States) before fixing for 1 h in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, United States). To decrease background fluorescence, cells were treated with 0.1% sodium borohydride (Sigma-Aldrich, St. Louis, MO, United States) in PBS for 10 min, washed, and then blocked with blocking buffer [10% normal goat serum (NGS) in PBS], and finally washed with 2% NGS in PBS (working buffer). Mouse anti-human FVIII (Bio-Rad Laboratories, Hercules, CA, United States) at a dilution of 1:1,000 and rabbit anti-human vWF (Abcam, Cambridge, United Kingdom) at a dilution of 1:100 in working buffer were added, and the devices were placed on a shaker for 20 min before then being placed at 4°C overnight. The devices were washed with working buffer, and donkey anti-mouse Alexa Fluor 594 and mouse anti-rabbit Alexa Fluor 488 secondary antibodies (Invitrogen, Carlsbad, CA, United States) diluted 1:400 in working buffer were added to the devices and incubated for 30 min on a shaker. After washing with PBS, DAPI at a dilution of 1:1,000 was added for 5 min, followed by an additional wash before being coverslipped and imaged. The cells were then imaged in representative areas of each device using an Olympus BX30 microscope with a 10X objective.