Anti human von willebrand factor

Tissues were embedded in OCT, frozen on liquid nitrogen, and 7 μm cryostat sections were cut. Specimens were placed on glass slides, air dried and fixed with aceton for 2 min at −20°C. After rehydration with 80% methanol at 4°C, the specimens were washed in PBS and then in PBS with 5% donkey serum and 1% bovine serum albumin, followed by incubation with the respective primary antibodies. Standard H&E and immunofluorescence stainings were performed as described previously [9] (link), [29] (link), using the following antibodies: anti-mouse LYVE-1 (AngioBio), biotin anti-MECA-32 (BD Biosciences), anti-human von Willebrand factor (Dako), anti-mouse podoplanin (clone 8.1.1, Developmental Studies Hybridoma Bank, University of Iowa), anti-mouse keratin 6, anti-loricrin (Covance Research Products), anti-BrdU-Alexa Fluor 594 (Invitrogen), anti-mouse F4/80 (AbD Serotec), anti-mouse CD68 (Abcam), anti-mouse CD8 (BD Biosciences) and anti-mouse CXCR4 (R&D Systems). Alexa Fluor488-, Alexa Fluor594- and Alexa Fluor647-coupled secondary antibodies and Hoechst 33342 were purchased from Invitrogen.

Paraffin-embedded colon sections were deparaffinized, rehydrated, subjected to antigen retrieval, and incubated with 3% H₂O₂ to eliminate endogenous peroxidase activity. Sections were incubated with 10% skim milk to reduce non-specific reactions and incubated overnight at 4°C with anti-rat CD68 (1:100; AbD Serotec, Kidlington, UK), anti-human α-smooth muscle actin (1:100; Dako Cytomation, Glostrup, Denmark), anti-human Von Willebrand Factor (1:100; Dako Cytomation, Glostrup, Denmark), anti-TNF-α (1:200; Novus Biologicals, Colorado, USA), anti-IL6 (1:100; Novus Biologicals, Colorado, USA), and anti-arginase 1 (1:200; Abcam, Massachusetts, USA). The samples were then incubated with horseradish peroxidase-conjugated polymer (HRP Polymer Conjugate, Invitrogen, CA, USA). Signals were detected by adding the chromogenic substrate AEC. Sections were rinsed with deionized water, counterstained with hematoxylin, and mounted for histological analysis.

Culture chamber slides were coated with fibronectin and seeded with ECFC and bmMPC at a 1:1 ratio. After in vitro co-culture for 7 days, bmMPC differentiate into VSMC/pericytes [43 (link)]. Once differentiated, cells were fixed with cold pure methanol on ice for 10 min. For immunostaining of ECFC, samples were incubated with a mAb anti-human von Willebrand factor (Dako) for 1 h at room temperature, followed by incubation with the secondary antibody Texas Red anti-mouse IgG (Vector) for 1 h at room temperature. Differentiated bmMPCs were incubated with anti-human calponin (Abcam), anti-human Sm22α (Abcam), anti-PDGFRb (Santa Cruz), anti-human NG2 (R&D Systems), anti-human αSMA (Sigma) or a negative control antibody (Santa Cruz). After washing samples were incubated with the appropriate FITC-labeled secondary antibody, and mounted using Vectashield with DAPI (Vector).