Von willebrand factor

The following primary mouse anti-human protein antibodies (clone; manufacturer)
were used in this study: RORγt (6F3.1; Merck Millipore, Darmstadt, Germany), CD3
(A-1; Santa Cruz Biotechnology, CA), CD20 (L26; Immunologic, Duiven,
Netherlands), CD79α (JCB117; Dako, Glostrup, Denmark), and Podoplanin (D2-40;
Dako). The following rabbit anti-human protein antibodies were used: T-bet/Tbx21
(EPR9302; Abcam, Cambridge, UK), GATA3 (EPR16651; Abcam), CD3 (SP7;
Immunologic), CD56 (MRQ-42; Cell Marque, Rocklin, CA), and von Willebrand Factor
(polyclonal; Dako).
As secondary reagents we used alkaline phosphatase (AP)- or horseradish
peroxidase (HRP)-conjugated anti-mouse and anti-rabbit BrightVision detection
kits (Immunologic). Chromogens PermaBlue/AP, PermaRed/AP, and PermaGreen/HRP
were from Diagnostic Biosystems (Sanbio, Netherlands), Deep Space Black was
purchased from Biocare Medical (Concord, CA) and Nova-RED was from Vector
Laboratories (Burlingame, CA). Antibodies and polymers were always diluted in
Normal Antibody Diluent from Scytek Laboratories (Logan, UT). We used
Tris-buffered saline solution with 0.05% Tween (TBST) as washing buffer between
incubation steps. The stained sections were mounted in Glycerol Gelatin aqueous
slide mounting medium (Sigma-Aldrich, Zwijndrecht, Netherlands) or in
Pertex^TM (VWR international, Amsterdam, Netherlands).

The middle parts of explanted TEVG samples were fixed in 10% formalin for 24 hours at 4°C, and then embedded in paraffin for standard histologic analysis with hematoxylin and eosin, Masson's trichrome, Verhoeff-Van Gieson (VVG), and von Kossa (VK) staining. For VK staining, human placenta tissue was used as a positive control. For immunohistochemistry, the tissue sections were deparaffinized, rehydrated, and blocked for endogenous peroxidase activity and nonspecific staining. The primary antibodies used included von Willebrand Factor (1:2000; Dako, Glostrup, Denmark), α-smooth muscle actin (1:500; Dako), and CD68 (1:200; Abcam, Cambridge, UK). Biotinylated secondary antibodies and streptavidinated horseradish peroxidase were then used before the color development of chromogenic reaction with 3,3-diaminobenzidine (Vector Laboratories, Burlingame, Calif). Nuclei counterstaining was performed with Gill's hematoxylin (Vector Laboratories).

Cells were seeded in 96-well plates (Greiner, Frickenhausen, Germany) and cultured until at least 80% confluence was reached. After fixing with ice-cold methanol (VWR, Darmstadt, Germany) and blocking with 5% normal goat serum (NGS; Dako, Glostrup, Denmark) in 0.1% Triton-phosphate-buffered saline (Sigma-Aldrich), the cells were stained with anti–α-SMA monoclonal antibody (dilution 1:1000; Sigma-Aldrich) and von Willebrand factor (dilution 1:200; Dako) for 1 h at 37°C. Subsequently, they were incubated for 1 h at 37°C with rhodamine-conjugated goat-anti-mouse and goat-anti-rabbit secondary antibodies (Alexa Fluor 594, 1:400; Invitrogen). Nuclei were counterstained with DAPI nucleic acid stain (Molecular Probes). As negative controls, samples were incubated with the secondary antibody only.
The OCA cells exhibited abundant α-SMA expression (Fig. 3A), while the OUA cells were mostly quiescent fibroblasts containing no α-SMA (Fig. 3B). Endothelial phenotype was excluded by the absence of von Willebrand factor.