Fig. 4
Localization and expression of Giardia α-SNAPs in trophozoites, encysting trophozoites and cysts. a Immunofluorescence localization, with polyclonal antibodies, of α-SNAP17224 (left column), α-SNAP16521 (middle column) and α-SNAP10856 (right column) in trophozoites, encysting trophozoites (8, 16 and 48 h post-induction of encystation) and cysts. The caret marks the perinuclear region, the arrowhead marks the PDR, and the triangle indicates PVs. To show the localization of α-SNAP16521 at both the PDRs and the ventral disc periphery (48 h post-induction), the corresponding panel is an overlay of two z-sections (individual images of the z-stack shown in Additional file 1: Figure S5d). Inset depicts overlay of the DIC and DAPI images. b Expression of the α-SNAP genes in trophozoites, encysting trophozoites and cysts were determined by real-time PCR (lower panel), where the expression of CWP1 gene serves as a positive control (upper panel). The asterisks indicate the significance of the difference between the expression under a given condition with that in trophozoites (**P < 0.01; ***P < 0.001; ns, not significant). c Colocalization of α-SNAP17224 and α-SNAP10856 in 48 h encysting trophozoites (top row) or that of α-SNAP16521 and α-SNAP10856 in trophozoites (bottom row). Insets depict magnification of the region of interest (ROI) that has been marked with a white box. The scattergram in each row indicates the analysis of colocalization between the two fluorophores over the entire z-stack by considering all the pixels within the whole area occupied by that cell. The values for Pearson correlation coefficient (Pr) and overlap coefficient (OC) written inside the scattergrams. The intensity plots at the extreme right indicate changes in fluorescence intensity of the red and green signals across the dotted white line in the ROI. d Mean Pr and OC values for multiple z-stacks to determine the extent of colocalization of either α-SNAP17224 or α-SNAP16521 with α-SNAP10856. Scale-bars: a, c, 5 μm