Anti fibrinogen antibody

Tissue sections (10 μm) of the paraffin-embedded PVA sponges were de-waxed and incubated with citrate buffer (0.01 M Citric Acid, 0.05% Tween 20, pH 6.0) for 20 minutes at 95°C for antigen retrieval. After quenching endogenous peroxidase enzymatic activity with 3% hydrogen peroxide and blocking endogenous biotin and binding sites as described by the manufacturer (Vector Labs, Burlingame, CA), the sections were subjected to immunostaining with anti-fibrinogen antibody (DAKO, Carpenteria, CA) and detection with an anti-rabbit Vectastain kit (Vector Labs, #PK-4001). Similarly, hCD45⁺ cells were detected with anti-CD45 antibody (BD Biosciences) using a Vectastain kit and DAB detection. Exposure-matched micrographs of immunostained slides were captured with an FSX100 microscope (Olympus America, Center Valley, PA).

Various concentrations (0. 1 – 100 μM) of compounds were plated in white 384-well plates (Greiner) and were incubated with 10 nM biotinylated Aβ42 (Anaspec) and 1 nM fibrinogen for 30 min at room temperature (RT) in a final volume of 10 μl assay buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 0.1% BSA). The mixture was then incubated with anti-fibrinogen antibody (Dako), 20 μg/ml streptavidin-conjugated donor, and protein A-conjugated acceptor beads (PerkinElmer) for 90 min at RT. Samples were read by a PerkinElmer EnVision plate reader. RU-505, which is a known inhibitor of Aβ42-fibrinogen interaction^{27 (link)}, was used as a positive control and also for comparison of its efficacy with other compounds. All the experiments were performed in triplicate. To determine IC₅₀ of compounds, the data were fitted to a sigmoidal dose-response equation (Y= Bottom + (Top − Bottom)/1 + 10^{(logIC50 − x) Hill coefficient)}) using GraphPad Prism 4 to calculate IC₅₀.