Complement profile in serum/plasma samples and immunofluorescence analysis of the wall of the occluded femoral artery for the deposition of β₂GPI and complement components. (A) CH50 hemolytic activity was evaluated in serum samples collected from the patient before (February 1, 2015) and at various time points after the infusion of eculizumab. A pool of sera from 25 healthy blood donors (normal healthy sera [NHS]) was used as control. The levels of C5a and soluble C5b-9 were measured in plasma samples at the same time points. A pool of plasma from 25 healthy blood donors (normal healthy plasmas [NHP]) was used as control. (B) The tissue was stained for β₂GPI with biotin-labeled recombinant antibody MBB2ΔCH2 followed by phycoerythrin-labeled streptavidin. To evaluate deposition of human IgG, CY3-labeled goat anti-human IgG was used; goat polyclonal antibodies were used to detect C1q, C4, and C3 followed by tetramethylrhodamine-labeled rabbit anti-goat IgG. C9 deposition was revealed by mouse monoclonal antibody (aE11) and CY3-labeled goat anti-mouse IgG. von Willibrand factor (vWF) on tissue was detected by using polyclonal rabbit IgG followed by fluorescein isothiocyanate–labeled horse anti-rabbit IgG. The sections were examined under a Leica DM2000 fluorescence microscope (Leica) equipped with a digital camera (DFC 490; Leica). Images were acquired by using Leica Application Suite Software. Original magnification ×200. Note the deposition of β₂GPI, IgG, C1q, C4, and C3, double-stained together with vWF on the endothelium, whereas C9 was predominantly localized in the subendothelium region.