pHrodo whole mount phagocytosis assay

3 dpf mpx:GFP transgenic zebrafish embryos were injected with 1 nL of 1 mg/ml pHRODO E. coli Red particles (Invitrogen). Systemic delivery was achieved by microinjection into the duct of Cuvier by two 0.5 nL injections at 12 psi. Embryos were then incubated in the dark for 2 hr before imaging in low melt agarose using a confocal microscope (Zeiss LSM800). Analysis was performed using MATLAB to filter for Red pHRODO beads within GFP+ neutrophils only and then using global thresholding to measure area and mean intensity of the pHRODO beads (Mathworks).