Aquatic organisms have specialized cells called ionocytes that regulate the ionic composition, osmolarity, and acid/base status of internal fluids. In small aquatic organisms such as fishes in their early life stages, ionocytes are typically found on the cutaneous surface and their abundance can change to help cope with various metabolic and environmental factors. Ionocytes profusely express ATPase enzymes, most notably Na⁺/K⁺ ATPase, which can be identified by immunohistochemistry. However, quantification of cutaneous ionocytes is not trivial due to the limited camera’s focal plane and the microscope’s field-of-view. This protocol describes a technique to consistently and reliably identify, image, and measure the relative surface area covered by cutaneous ionocytes through software-mediated focus-stacking and photo-stitching–thereby allowing the quantification of cutaneous ionocyte area as a proxy for ion transporting capacity across the skin. Because ionocytes are essential for regulating ionic composition, osmolarity, and acid/base status of internal fluids, this technique is useful for studying physiological mechanisms used by fish larvae and other small aquatic organisms during development and in response to environmental stress.

Here we describe the immunolocalization of a membrane-bound proton pump, the V-type H⁺-ATPase (VHA), in tissues and isolated cells of scleractinian corals. Immunolocalization of coral proteins requires additional steps not required for various model organisms, such as decalcification of the coral skeleton for immunohistochemistry or removal of cells away from the skeleton for immunocytochemistry. The tissue and cell preparation techniques described here can be adapted for localization of other coral proteins, provided the appropriate validation steps have been taken for the primary antibodies and species of coral used. These techniques are important for improving our understanding of coral cell physiology