Application suite software las v3.8

Fish were anesthetized for imaging by placing them in ice-cold system water for approximately 15 seconds. Fish were mounted on a 2% agarose bed at 35x magnification under the GFP (488nm) fluorescent filter, and imaged on a Leica stereomicroscrope (M205FA) using Leica Application Suite software (LAS v3.8; Leica, Wetzlar, Germany). Images were collected with varying exposure levels (300ms–600ms) owing to slight differences in calcein and DASPEI staining sensitivity per individual. After lateral images were collected from the right and left sides of the face, the fish was measured for SL and revived in system water.

Live fish were stained with calcein (Sigma Aldrich C0875) to label bone and 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI; Sigma Aldrich D3418) to label canal (CN) and superficial neuromasts (SN) as described in Powers et al. (2018b) (link). For imaging, fish were anesthetized by immersion in ice-cold system water for approximately 15 seconds. Fluorescent images were collected at 7.81x magnification under the GPF (calcein; 488nm) and TXR (DASPEI; 561nm) filters using a Leica stereomicroscope (M205FA) and Leica Application Suite software (LAS v3.8, Wetzlar, Germany). Images were overlaid to visualize bone and neuromasts (Figs. 1, 3 and 5).