Fig. 4. Expression of AeNHE3, Na⁺/K⁺-ATPase and V-ATPase in the Malpighian tubules and hindgut of Aedes aegypti. Whole-mount (A,G) or paraffin sections (B-F) were analyzed by labeling with V-ATPase B-subunit (A,B), anti-NHE3 antibody (D-G) and anti-Na⁺/K⁺-ATPase 1 subunit monoclonal antibody (E-G). Preimmune serum was used as negative control (C) with phalloidin (green), and similar results were obtained with peptide preabsorbed anti-NHE3 antibody. V-ATPase is primarily localized to the apical membrane of principal cells (PC in A,B) whereas both NHE3 (D,F) and Na⁺/K⁺-ATPase (F) localize to basal aspect of the plasma membrane of principal cells. In median Malpighian tubules NHE3 expression was also detected in the apical membrane (E, arrow) and in the basal membrane colocalizing with Na⁺/K⁺-ATPase (E, arrowhead). Weaker expression of NHE3 was also detected as punctate structures in the cytoplasm of Malpighian tubules (F,F1). Anterior hindgut and Malpighian tubules exhibit similar expression levels of NHE3, whereas in the rectum both NHE3 and Na⁺/K⁺-ATPase are remarkably enhanced (G,G1,G2). E1,F1,G1 show NHE3 expression (red), while E2,F2,G2 depict Na⁺/K⁺-ATPase expression (yellow), and merged channels depicting co-localization of NHE3 and Na⁺/K⁺-ATPase are shown in E-G. Scale bars, 50 µm (A-F); 100 µm (G).