Dig digoxigenin rna labeling kit

Whole-mount in situ hybridization was performed as described previously [34 (link)]. The liver-type fatty acid-binding protein 10a (fabp10a) and hepatocyte nuclear factor 3-gamma (foxa3) were amplified by PCR reaction with KOD Dash (TOYOBO) with the primer sets listed in Table S1. This was followed by subcloning to the pTAC-2 TA vector (BioDynamics) and in vitro transcription with SP6 RNA polymerase (New England Biolabs, Ipswich, MA, USA) with the DIG (digoxigenin) RNA Labeling Kit (Sigma-Aldrich) to obtain antisense RNA probes. Four percent paraformaldehyde (PFA)-fixed embryos were hybridized with digoxigenin-incorporated RNA probes at 65 °C overnight. Following hybridization and washing, the embryos were incubated with an anti-DIG antibody conjugated with alkaline phosphatase (Sigma-Aldrich) at 4 °C overnight. Color reaction was performed via incubation in NBT/BCIP Ready-to-Use Tablets substrate (Sigma-Aldrich).

Whole-mount in situ hybridization was performed as described previously [52 (link)]. Paraformaldehyde-fixed embryos were hybridized with digoxigenin-incorporated antisense RNA probes of CYP subtypes at 65 °C overnight (DIG (digoxigenin) RNA Labeling Kit, Sigma-Aldrich). To obtain the CYP probes, CYP DNAs were amplified by PCR reaction with KOD FX Neo enzyme solution (Toyobo) with the primer sets listed in Table S2. This was followed by subcloning to pTAC2 TA vector (BioDynamics, Tokyo, Japan) and in vitro transcription with SP6 RNA polymerase (New England Biolabs, Ipswich, MA, USA). Following hybridization and washing, the embryos were incubated with anti-DIG antibody conjugated with alkaline phosphatase (Sigma-Aldrich) at 4 °C overnight. The color reaction was performed by incubation in BM Purple substrate or Fast Red (Sigma-Aldrich).