Adult worms, metacercariae and eggs of C. sinensis were homogenized in lysis buffer (40 mM Tris, 7 M urea, 2 M thiourea and 4% CHAPS), respectively. The concentration of each crude extract was determined by the Bradford assay (Bio-Rad Laboratories, Berkeley, CA, USA). A 10-μg sample of each crude extract was subjected to 12% SDS-PAGE and electrotransferred to a polyvinylidene fluoride membrane. The anti-CsTegu20.6 serum (1:1000 dilution) and HRP-conjugated anti-rat IgG (1:2000 dilution) were used primary and secondary antibodies, respectively. Final visualization was performed using the ChemiDocTM imager (Bio-Rad).