Goat anti rabbit antibody conjugated to 10 nm gold particles

5 µL droplets of semen or pre-formed SEVI fibrils were placed on a Formvar-coated copper grid (Ted Pella Inc.) and incubated for 5 min. The grid was washed three times (10 s each) with 50 µL droplets of PBS, pH 7.4 (1x Dulbecco’s PBS, Sigma Aldrich). Excess solvent was carefully soaked off with filter paper, before a 50 µL droplet of 5 % bovine serum albumin in PBS was applied for 15 min. Afterwards the grid was washed with a droplet of 50 µL buffer. Excess solvent was removed with filter paper and the grid was incubated for 1 h with either 50 µL of anti-SEVI antiserum or respective pre-immune serum (both diluted 1:100 in PBS) and covered by a Petri dish. The grid was washed in PBS as described above, before a 50 µL droplet of goat anti-rabbit antibody conjugated to 10 nm gold particles (Sigma Aldrich, 1:100 dilution in PBS) was applied. The grid was then washed again with a 50 µL droplet of PBS containing 0.05 % Tween 20 for 20 s and three droplets of 50 µL water for 10 s each. Finally, the grid was stained using a series of three 50 µL droplets containing 2 % uranyl acetate (20 s each), and then dried with filter paper. All steps were carried out at room temperature. Semen samples labelled with anti-SEVI antiserum and respective pre-immune serum were examined under a Zeiss 900 electron microscope, operated at an acceleration voltage of 80 kV.