Immunofluorescence microscopy

Samples of outer mantle and inner mantle were excised and immersed in 3% paraformaldehyde diluted in seawater at 4°C overnight. Sample preparation and immunostaining were performed following the method of Hiong et al. (2017b) except that the antigen retrieval was carried out using 0.05% citraconic anhydride with heating at 90°C for 10 min, and the concentration of the custom‐made anti‐CA2 rabbit polyclonal antibody (Genscript) used was 1.67 μg mL⁻¹.

After immunostaining, sections were viewed under an Olympus BX60 epifluorescence microscope (Olympus Corporation, Tokyo, Japan) mounted with an Olympus DP73 digital camera (Olympus Corporation) for image capturing. All images were captured under predetermined optimal exposure settings. Of note, zooxanthellae are known to contain pigments that autofluoresce (Castillo‐Medina et al. 2011), therefore, they were visualized using a U‐MWIG filter (Olympus) with excitation at 520‐550 nm and 580 nm band‐pass emission filter (red channel). CA2‐like immunostaining was observed using the U‐MNIBA filter (Olympus) with excitation at 470–490 nm and 515–550 nm band‐pass emission filter (green channel). Corresponding differential interference contrast (DIC) images were captured for tissue orientation.