Intracellular Ca2+ Imaging using Fluo-4 AM

Cell-permeable Ca²⁺ indicator Fluo-4 AM (cat. no. F14201; Invitrogen; Thermo Fisher Scientific, Inc.) were used to monitor levels of intracellular Ca²⁺, based on the literature (36 (link)). Following immersion in HBSS without Ca²⁺ and Mg²⁺ for 20 min at 20-24°C, the organ explants were immersed in dye (cat. no. F14201; Invitrogen; Thermo Fisher Scientific, Inc.) for 25 min following Fluo4-AM loading at 24°C. Subsequently, the explants were fixed in 4% paraformaldehyde for 30 min at 20-24°C. Phalloidin (1:200) was applied for 1 h at 20-24°C and protected from light. For measurement of Fluo-4 Ca²⁺ signals, organs were imaged under a BX41 microscope at ×400 magnification (Olympus Corporation) with epifluorescence, and the images were obtained using a TCS-SP5II laser-scanning confocal micro-scope (×400 magnification; Leica Biosystems GmbH) in 20 consecutive fields.