Metal enhancer for dab stain

Whole-mount immunostaining was performed as described (Suga et al., 2006 (link); Yasuoka et al., 2009 (link); Yasuoka et al., 2014 (link)) with modifications for fluorescent imaging. Briefly, embryos were bleached before staining. 5D4 antibody (mouse monoclonal IgG) was used as the primary antibody (Cosmo Bio, PRPG-BC-M01, 1/100 diluted). HRP-conjugated anti mouse IgG (Promega, 1/500 diluted) or AlexaFluor488-conjugated anti-mouse IgG (Thermo Fisher Scientific, A-11001, 1/200 diluted) was used as the secondary antibody. For HRP staining, a Peroxidase Stain DAB Kit and Metal Enhancer for DAB Stain (Nacalai Tesque) were used. Automated immunolabelling experiments were performed with InsituPro VSi (Intavis). Some DAB-stained embryos were subjected to cross-sections stained with hematoxylin. Stained embryos were observed under a fluorescence microscope (Leica M205 FA or Keyence BZ-X810).

The virus stock was diluted 10-fold (1:10–1:10⁶) in FBS-free minimal essential medium (MEM). Diluted viruses (250 μL) were inoculated into the Vero cell monolayer in a 24-well plate and incubated at 37 °C for 2 h. The cells were overlaid with 500 μL MEM (Thermo Fisher Scientific, Cat #11935046) supplemented with 3% FBS and 1.5% carboxymethylcellulose sodium salt (Sigma-Aldrich, Cat# C4888-500G), and the plate was incubated at 37 °C for three days. The cells were washed three times with phosphate-buffered saline (PBS) (+) and fixed with 10% formaldehyde neutral buffer solution (Nacalai Tesque, Cat# 37152-51) for 20 min. After permeabilization with 1% Triton X-100 (Nacalai Tesque, Cat# 35501-15) in PBS (−) for 5 min, cells were incubated with mouse anti-flavivirus NS3 monoclonal antibody (34B1) [60 (link)] at 37 °C for 60 min. After washing with PBS (−), cells were incubated with goat anti-mouse IgG (H+L)-HRP (KPL, Cat# 074-1806) at 37 °C for 60 min. The foci of the infected cells were visualized using a Peroxidase Stain 3,3′-diaminobenzidine (DAB) Kit (Nacalai Tesque, Cat# 25985-50) prepared in the Metal Enhancer for DAB Stain (Nacalai Tesque, Cat# 07388-24).