Fig. 6. Immunohistochemistry was performed to detect AgCA9 and Na⁺/K⁺-ATPase protein in longitudinal sections of whole larvae embedded in paraffin. The images were generated on a laser confocal microscope (Leica SP2). The anterior end of the larvae is towards the left. B–D are images of the same section with the three different fluorochromes (FITC = green = AgCA9; TRITC = red = Na⁺/K⁺-ATPase; Cy5 = blue = DRAQ-5 nuclear stain, respectively). AgCA9 protein detection was variable in the epithelial cells of the GC but was consistently seen in the proteinaceous matrix that fills the GC lumen and lines the alimentary canal within the ectoperitrophic space (ectoperitrophic fluid) (A). Na⁺/K⁺-ATPase was used as a counter-stain to identify the regions of the alimentary canal (A,C,D). Na⁺/K⁺-ATPase is localized to the apical membrane in the cells of the AMG (C, open arrow) and to the basal membrane in the cells of the PMG (C, arrow). In the vicinity of the switch from apical to basal localization is the beginning of the TR (A, arrow; C, arrowhead). AgCA9 detection in the transitional region reveals an association with the periphery of the nucleus (B,D; e.g. arrows). Nuclear localization was confirmed by counterstaining with the nuclear stain DRAQ-5 (D, blue). For abbreviations, see Fig. 2 legend. Scale bars: 150 µm (A); 172.2 µm (B–D).