Goat antihuman ve cadherin

Human pulmonary microvascular endothelial cells (hPMVEC; PromoCell, Heidelberg, Germany), used at passages 4–6, were grown to confluence on glass slides, incubated with VT (300 ng/ml) or sterile PBS as a control for 90 minutes and then stimulated with LPS (0.1 μg/ml) from Escherichia coli (Sigma-Aldrich, Steinheim, Germany), PLY (0.25, 0.5, 0.75 μg/ml) [34 (link)], or PBS. After 30 minutes, cells were fixed in 3% formalin, and the tight junction protein vascular endothelial (VE)-cadherin (1:400, goat antihuman VE-cadherin; Santa Cruz Biotechnology, Santa Cruz, CA, USA; secondary antibody 1:8000, Alexa Fluor 488 F(ab′)₂ rabbit antigoat IgG (H + L), Invitrogen, Darmstadt, Germany) and actin fibers (1:2000; phalloidin Alexa 546-labeled; Invitrogen) were stained to evaluate cell morphology [35 (link)]. 4′,6-diamidino-2-phenylindole staining (Sigma-Aldrich) was used to visualize cell nuclei. The immunofluorescence of the cells was analyzed by confocal microscopy using an LSM 780 microscope with ZEN 2011 software (objective: Plan Apochromat × 63/1.40 oil; Carl Zeiss Microscopy, Jena, Germany).

Rabbit anti-mouse lyve-1 antibody (RDI-103PA50) was from Research Diagnostics Incorporated (Concord, MA). Donkey anti-rabbit and rat IgGs conjugated with alexa 488, 594 were from TebuBio (TebuBio, Le Perray en Yvelines, France). Anti-pSer473Akt, anti-Akt, anti-pErk, anti-Erk1/2 are from Cell Signalling Technology. Anti-Phalloidin was from Cytoskeleton (actin-stain phalloidin 488, cat PHDG1). Anti-phospho-VEGFR-3 is from Cell applications Inc. Rabbit anti-human LC3B, anti-human Notch/NCID and anti-p62 were from Cell Signaling (#2775), Goat anti-human VE-Cadherin from Santa-Cruz ((C19):SC6458) and Rabbit anti-human ATG5 and ATG7 from Sigma. Anti-human active beta-catenin was from Milliopore.

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.