Macrophage Visualization in Zebrafish

Neutral red staining was used to observe the macrophage, following published procedures (Herbomel et al., 2001 (link); Davis and Ramakrishnan, 2009 (link); Shiau et al., 2015 (link)). The 3 dpf zebrafish were wounded consistently by tailfin transection with a sterile scalpel. Subsequently, zebrafish were immersed in neutral red solution (2.5 μg/mL + 0.003% PTU) (Sigma Aldrich) at 28°C in the dark for 7–9 h. Furthermore, in order to observe the response of macrophages against mycobacterial infection, Mm were injected into the hindbrain of 30–32 hpf zebrafish embryos with 50 CFU according to the previous method (Gutzman and Sive, 2009 (link)). Zebrafish were then incubated in neutral red solution as already described. Anesthetized zebrafish were embedded into solidified agarose drops and viewed under light microscopy to acquire images (SteREO Discovery.V20; Carl Zeiss, Oberkochen, Germany).