Fig. 4. Immunofluorescence localization of Na⁺/K⁺-ATPase and V-ATPase. (A,E,I) Labelling with OregonGreen–phalloidin (green) reveals the organization of the apical domain of the secretory cells. (A) An optical cross-section through an entire salivary gland and (E,I) two horizontal planes through a gland as indicated by broken lines in (A). The apical surface of the secretory cells is involuted deeply into the epithelium to form a branching system of canaliculi (arrows) that embrace the DAPI-stained nuclei (blue in E,I). (B,F,J) Double-labelling of a longitudinal section through a salivary gland with antibody 5 against Na⁺/K⁺-ATPase -subunit (red) and phalloidin (green); (C,G,K) a longitudinal section co-stained with an antibody against V-ATPase subunit E (red) and phalloidin (green); (D,H,L) a cross-section co-labelled with an antibody against V-ATPase subunit d (red) and phalloidin (green); (J,K,L) composite images of phalloidin and antibody labelling. The basolateral domain but not the apical domain of the plasma membrane is intensely labelled for Na⁺/K⁺-ATPase. Antibodies against the various V-ATPase subunits stain the luminal side of the cell and the canaliculi (arrows). Moreover, some vesicular structures within the secretory cells are labelled by V-ATPase antibodies (yellow arrowheads in C,D,K,L). Immunofluorescence associated with the basal lamina (white arrowheads) surrounding the epithelial tubule is due to nonspecific binding of secondary antibodies. Scale bars, 20 µm.