Fig. 4. The specificity of the affinity-purified antibody 5563 was verified using a western blot (A) and immunohistochemistry (B–E). Part A shows the results of an antibody adsorption assay. The left panel of the image is the Fast Green staining of total protein to ensure equal loading. Antibody was pre-incubated with peptide prior to detection of protein. The western blot illustrates the detection of protein with pre-immune IgY (lane 1), affinity-purified antibody 5563 IgY (lane 2) and peptide-blocked IgY (lane 3). Images B–E illustrate immunohistochemistry results obtained using affinity-purified antibody 5563 IgY (E) and peptide-blocked IgY (B,C,D) in longitudinal sections of whole larvae embedded in paraffin. The images were generated on a laser confocal microscope (Leica SP2). B and C are images of the same section of larval alimentary canal (without the rectum) with two different fluorochromes (FITC = green = AgCA9; TRITC = red = Na⁺/K⁺-ATPase). D and E are images of the rectum positioned such that the anterior end is to the left. Na⁺/K⁺-ATPase immunostaining was used as a counter-stain to better visualize the regions of the alimentary canal in images C–E. The pre-adsorbed antibody (B–D) failed to detect AgCA9 protein compared with antibody not treated with peptide (E; also Fig. 6A). Apparent exoskeleton staining is non-specific (B,C; arrows). For abbreviations, see Fig. 2 legend. Scale bars, 150 µm (B,C); 75 µm (D,E).