Hrp conjugated anti mouse secondary antibody sc2005

VWF and recombinant AIM-A1 fragments (6 μg/mL in PBS) were coated to high-binding half-area 96-well plates (Corning). VHH81 binding to immobilized VWF and AIM-A1 fragments were detected with horse-radish peroxidase (HRP)-conjugated anti-VHH monoclonal antibody 96A3F5 (Genscript) (1:1000). Purified GPIb-IX complex was coated to the plate using 0.1% Triton X-100, 20 mM Na₂CO₃/NaHCO₃, pH 9.6. Fixed platelets, prepared from human washed platelets followed by two rounds of washing in 4% paraformaldehyde, were coated to the plate using 1% poly-l-lysine (Sigma) in PBS. Bound AIM-A1 fragments were detected with HRP-conjugated anti-HisTag antibody 4E3D10H2/E3 (1:2000). Monoclonal antibody binding to AIM-A1 fragments was detected using HRP-conjugated anti-mouse secondary antibody sc2005 (Santa Cruz Biotechnology) (1:2000). In all cases, plates were washed three times with HEPES buffered saline with 0.1% Tween-20 on a BioTek ELx405 plate washer. After binding and washing, 1-Step Ultra-TMB substrate (ThermoFisher) was added to each well, quenched with 2 M H₂SO₄. Absorbance was measured at 450 nm. Empty wells were used to subtract baseline absorbance.