Analysis of C3 Cleavage in Vaginal Lateral Fornix and External Cervix Samples

Western blot analyses were performed using in-house protocols (34). The samples were thawn on ice, centrifuged 12,000 × g for 3 min and diluted 1:10 in sterile PBS. 20 µl portions of a dilution series of samples (in PBS) in SDS and 5% mercaptoethanol containing sample buffer were loaded onto 4%–12% SDS-PAGE gels and run under reducing conditions. A normal human serum (NHS) pool was obtained from healthy laboratory personnel after a written informed consent and used as a reference. The proteins from the gel were electrotransferred to a nitrocellulose filter. To prevent nonspecific binding the nitrocellulose membranes were incubated in 5% milk in PBS/Tween 0.05% for 1 h. The membranes were then incubated with rabbit anti-human C3c antibody (Dako; final dilution: 1:10,000 in milk/PBS/Tween) overnight at +4°C. For additional detection of C3-IgG complexes, also rabbit antibodies against C3d (Dako) were used similarly as anti-C3c antibodies. The membranes were washed with PBS/Tween and incubated for 1 h at RT with HRP-goat-anti-rabbit IgG antibody (Jackson ImmunoResearch; 1:10,000 in milk/PBS/Tween). Finally, the membranes were washed, and protein bands were visualized by electrochemiluminescence.