Fig. 3.

Localization of SmGPCR in larval stages of S. mansoni. Samples were fixed with 4% PFA or ice-cold acetone, permeabilized with 0.5% Triton X-100 and then probed with anti-SmGPCR IgG followed by a FITC-labeled 2ry antibody. Red TRITC-phalloidin was used as a counterstain to visualize the musculature. Sporocysts: SmGPCR immunofluorescence (green) was detected on the tegument (arrows) and within the parenchyma of 4-day-old in vitro cultured sporocysts incubated with anti-SmGPCR (A and B) but not the pre-adsorbed anti-SmGPCR IgG control (C and D). An overlay of the phalloidin (red) and SmGPCR (green) signals showed no apparent co-localization (B). Schistosomula: In vitro transformed schistosomula were cultured for 4 days (E) or 8 days (G) and then probed with anti-SmGPCR antibody. Fluorescence can be seen in the acetabulum, the tegumental region, the esophagus and parenchyma cells. A close-up of the posterior end of a 8-day animal shows a distinct pattern of SmGPCR fluorescence in the acetabulum (H). No significant immunoreactivity could be detected in the negative controls treated with il3-preadsorbed antiserum (F), pre-immune serum or when the primary antibody was omitted (not shown). 28-day-old schistosomula were probed with anti-SmGPCR antibody (green) and phalloidin (red) to test for possible colocalization of the receptor with the musculature. Red phalloidin labeling of the major longitudinal, circular and oblique body wall muscles is clearly visible (I). The overlay identified significant co-localization (yellow fluorescence) in the muscle cone of the head region (arrow), musculature of the acetabulum (arrow head) and the subtegumental musculature (box) (I and J). A close-up of the parasite body wall shows co-localization of SmGPCR and phalloidin in the outer layer of the musculature (yellow fluorescence) (K).