Mzfliii

Adult guts were dissected in PBS and fixed in 4% paraformaldehyde for 45 mins at room temperature; wing discs from 3^rd instar larvae were fixed in 4% paraformaldehyde for 20 mins at room temperature. Tissues were washed with PBS + 0.1% Triton X-100 and blocked with PBS + 0.1% Tween-20 + 10% BSA for 1 h at room temperature. The samples were incubated with primary antibody (diluted in PBS + 0.5% Triton X-100) at 4 °C for 1–3 days. Secondary antibody incubation was carried out at room temperature for 2 h. The samples were subsequently stained with DAPI (2 μg/ml) and mounted in Prolong Gold Antifade Reagent (Invitrogen). Confocal images were captured on a Nikon A1RSi laser scanning confocal microscope, Nikon CSU-W1 spinning disk confocal microscope, or Nikon Yokogawa CSU-W1 SoRa spinning disk confocal microscope and processed with Adobe Photoshop / Illustrator software from Adobe Suite 2023. Adult wings were mounted in Mowiol and their images acquired using a Leica MZFLIII stereomicroscope with a Zeiss Axiocam 208 camera and Nikon Zen 3.0 software.

Adult fish were anesthetized and placed on a 1% agarose plate with the caudal fins spread out. Zebrafish embryos, larvae and juveniles were anesthetized in E3 embryo medium containing 0.1 mg/ml tricaine. The plate was placed under a Leica MZ FLIII dissection microscope and images were taken using the AxioCam HSM digital camera and AxioVision AC software (Carl Zeiss). For live confocal imaging, fish were anesthetized and immersed in 0.17mg/ml tricaine in a petri dish. The caudal fins were flattened to the bottom of the petri dish with a slide hold-down (Warner Instruments 64–0248) and imaged with a water-immersion objective equipped on Nikon A1RsiMP Confocal. All images were processed using ImageJ (NIH).