Mm 33

All imaging experiments were done on 3 dpf Tg(sspo:sspo-GFP), Tg(sspo:sspo-GFP;pkd2l1:tagRFP;pkd2l1:GCaMP5G), and Tg(sspo:sspo-GFP;β-actin:Arl13b-GFP) zebrafish larvae. Their respective siblings were used as controls (i.e., wild-type and heterozygous mutants from the same clutch). For all experiments, larvae were laterally mounted in glass-bottom dishes (MatTek, Ashland, Massachusetts, USA), filled with 1.5% low-melting point agarose. Unless otherwise noted, larvae were paralyzed by injecting 1–2 nL of 500 mM alpha-bungarotoxin (TOCRIS) in the caudal muscles of the trunk via glass micropipette held by a micromanipulator (Märzhäuser Wetzlar MM-33), using a pneumatic Pico-pump (World Precision Instruments PV-820).

Coronary flow reserve (CFR) was used as an index of coronary microvascular function and measured as previously described (Hartley et al., 2008 (link)). Briefly, after the animal was anesthetized with 1% isoflurane, a pulsed Doppler ultrasound probe tip was placed at the left side of its chest and stabilized by a micromanipulator (model MM3-3, World Precision Instruments). The probe was fixed in a position when peak coronary velocities were identified. As a coronary vasodilator stimulus, basal (with 1% isoflurane) and a dilated (induced with 2.5% isoflurane) coronary flow were recorded and analyzed using a Doppler ultrasound system (Indus Doppler flow velocity system). Ratios of peak velocities at dilated and a basal state were reported as CFR.