Goat anti human fibrinogen

Samples were boiled in SDS sample buffer, subjected to SDS-PAGE on 4% to 12% Bis-Tris polyacrylamide gels (NuPAGE; Invitrogen), and visualized using a Typhoon Trio variable mode imager system and ImageQuant software. CpaA was identified by liquid chromatography-mass spectrometry (LC-MS/MS) of gel-excised material containing A. baumannii culture supernatant (MS Bioworks, Ann Arbor, MI).
Samples were subjected to SDS-PAGE and immunoblotting using anti-human fXII (Molecular Innovations), 1:5,000 sheep anti-human fV (Molecular Innovations), and 1:5,000 goat anti-human fibrinogen (Sigma). Horseradish peroxidase-conjugated secondary antibodies (fXII [1:2,000], fV 1:10,000], and fibrinogen [1:1,000]; Jackson Laboratories) were used, and the blots were visualized with SuperSignal West Pico Substrate (Thermo Scientific). Developed blots were imaged using a Typhoon Trio imager (GE Healthcare). Rabbit anti-mouse fXII (Molecular Innovations) and Alexa Fluor 680-conjugated goat anti-rabbit antibodies were used for detection of mouse fXII. Blots were imaged by using the Odyssey imaging system.

Selective agonist blocking was achieved by incubating an upstream priming region with the appropriate antibody solution for 30 min prior to device assembly. Priming regions were isolated using a custom removable PDMS microwell to prevent the antibody contamination of the rest of the flow channel. Collagen I was blocked using rabbit antihuman collagen type I (both from Sigma Aldrich), von Willebrand Factor was blocked using sheep antihuman von Willebrand Factor (both from Haematologic Technologies Inc.), and fibrinogen was blocked using goat antihuman fibrinogen (Sigma Aldrich). The samples were rinsed three times with distilled deionized water following incubation, with care taken to not contaminate areas outside of the priming regions. Devices were then assembled, passivated with albumin as described above, and used immediately.