Fig. 5. Localization of carbonic anhydrase in branchial tissue of Riftia pachyptila. (A) Negative control with sense riboprobe on a transverse section of the plume showing its anatomical organisation. Lamellae are attached to the central obturaculum (Ob), and are composed of filaments (Fl) joined proximally but free distally. (B—D) In situ hybridization of CA mRNA with DIG-labeled riboprobe. (B) Transverse section of a single filament; the arrowhead indicates maximum staining intensity observed in a pinnule. (C,D) Epithelial cells are clearly stained by CA riboprobe, contrasting with the weak staining of extracellular matrix (C) and vascular vessels (D). (E) Negative control for CA immunolocalization using rabbit anti-chick CA II antiserum. (F) Corresponding protein immunolocalization restricted to the apical region of the epithelial cells. (G) Protein localization with DNSA, and (H) control section with DNSA on slides pre-incubated with the competitive CA inhibitor ethoxyzolamide. AV, afferent blood vessel; CF, coelomic fluid; EV, efferent blood vessel. Scale bar, 200 µm (A); 40 µm (E,G,H); 20 µm (B—D,F).

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