The tissue distribution of CsMRP4 in both adults and metacercariae was investigated via immunohistochemistry. The NBD1 region was chosen as the immune antigen for mice immunization, since it was more specific than NBD2 and the non-membrane-spanning region. With this strategy, we successfully produced and purified NBD1 of ABCC subfamily transporters . The 6× histidine-tagged rCsMRP4-NBD1 was then purified using Ni-NTA agarose (Additional file 8: Figure S5). The mouse anti-CsMRP4-NBD1 immune serum reacted well with rCsMRP4-NBD1 (24.9 kDa) by apparently detecting the native CsMRP4 in the crude extract of the adult worm (Fig. 5b). It was, therefore, applied to immunohistochemical staining. CsMRP4 was distributed mainly in the oral sucker and mesenchymal tissues of the adults (Fig. 6a, b) and metacercariae (Fig. 6c). Moreover, the ventral sucker of the metacercariae showed strong localization of CsMRP4.Fig. 6Localization of CsMRP4 in C. sinensis adults (a, b, d, e) and metacercariae (c, f) detected by immunohistochemistry. Top panels (a - c) were stained with mouse anti-NBD1 immune serum and bottom panels (d - f) were stained with normal mouse serum. Abbreviations: EB, excretory bladder; MT, mesenchymal tissue; OS, oral sucker; T, testis; VS, ventral sucker