Nitro blue tetrazolium 5 bromo 4 chloro 3 indolyphosphate substrates

Whole-mount RNA in situ hybridizations were performed as previously described (Nakamura et al., 2006 (link)). Fluorescein isothiocyanate (FITC)- and digoxigenin (DIG)-labeled probes were synthesized from the full-length medaka cDNA of ndrg1a, ndrg1b, and oct4 (stem cell marker also known as pou5f1; Wang et al., 2011 (link)) using pGEM-T Vector (Promega) linearized plasmid. Embryos at stage 35, and male brains and testis were fixed overnight in 4% RNAse-free paraformaldehyde at 4°C, permeabilized using 20 µg/µl proteinase K at room temperature (RT), and hybridized at 68°C overnight with either ndrg1a, ndrg1b, or oct4 DIG-labeled RNA probes, or at 66°C overnight with ndrg1b FITC-labeled RNA probe. Hybridized DIG probes were detected using an alkaline phosphatase–conjugated anti-digoxigenin antibody (1:2000; Roche) in the presence of nitro blue tetrazolium/5-bromo-4-chloro-3′-indolyphosphate substrates (Roche). Stained embryos were embedded in gelatin, cryostat sectioned at 14–16 µm thickness, and photographed. Hybridized FITC probes were examined under a fluorescence microscope.