Diphenyleneiodonium

Zebrafish at the prim25 stage (at approximately 36 hours post fertilization; staged according to the method of Kimmel, et al. (Kimmel et al., 1995 (link))), were manually dechorionated, and anesthetized in Tris-buffered tricaine methane sulfonate (Tricaine; 200 mg/ml; Western Chemicals, Inc., Frendale, WA) prior to injection. For injections, approximately 5 nL of opsonized polystyrene bead suspension at 1.5 × 10⁷ beads/ml in PVP, or 2 nL of C. albicans suspension at 1.0 × 10⁷ cfu/ml was microinjected through the otic vesicle into the hindbrain ventricle to achieve a dose of approximately 15 beads or 15 fungal cells at the site of injection. Within one hour post-injection, larvae were screened using a Zeiss Axiobserver Z1 microscope equipped with Vivatome system (Carl Zeiss Microimaging, Thornwood, NJ) for selection of larvae containing 13–17 beads/fungal cells at the site of injection (SOI). Larvae were subsequently incubated at 33°C in E3 media containing PTU. In experiments to test the relationship between extracellular fungal number and mortality, larvae were preincubated for 1 hour pre-infection with either DMSO or diphenyleneiodonium (Enzo, Farmingdale NY) as described (Brothers et al., 2013 ).