Figure 2.
Interactions of fibrinogen (Fg) with its receptors, cellular prion protein (PrPC) and intercellular adhesion molecule-1 (ICAM-1) on the surface of neurons detected by proximity ligation assay (PLA). Representative images of PLA signals shown as red dots indicating interactions between (A,B) Fg and PrPC (Fg-PrPC) and (C,D) between Fg and ICAM-1 (Fg-ICAM-1). Neurons were treated with medium alone (control) (A,C) or with 1 mg/mL of Fg (Fg1) for 24 h (B,D). Fg, PrPC and ICAM-1 were detected using anti-Fg, anti-PrPC, and anti-ICAM-1 antibodies, respectively. Enlarged (3× zoom) images (shown in yellow punctuated boxes) are pointed to by red arrows. Standardized size images of each selected area of interest were collected by acquiring at least 4 focal planes with increments of 0.5 µm to form a final z-stock image. Representative three-dimensional reconstructions of the PLA signals (Fg-PrPC and Fg-ICAM-1) on the surface of neurons from the respective z-stack images (Ba,Da). (E) One group of neurons treated with 1 mg/mL of Fg were probed with Immunoglobulin G (IgG) (Fg-IgG) and used as a negative control to define the specificity of the PrPC and ICAM-1 detecting antibodies. Cellular nuclei were labeled with 4′,6-diamidino-2-phenylindole shown in blue in all images. (F,G) Summary of data analysis of interaction of Fg with PrPC and ICAM-1, respectively. The number of PLA signals per cell was quantified into 7 randomly selected similar size areas of interest in each image and averaged for each treatment group. (H,I) Neurons were treated similar to B,D using PLA to detect an interaction of Fg with PrPC (H) and with ICAM-1 (I) with the addition of receiving counterstaining with microtubule-associated protein 2 (green) as a neuronal marker. Cellular nuclei were labeled with 4′,6-diamidino-2-phenylindole (blue). Hirudin was present in all experimental groups to prevent the conversion of Fg to fibrin. p < 0.05 in all; ∗—vs. Control; n = 4.