RNAscope analysis was performed using RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics #323100) with a few modifications to the previously described method by Nomaru et al. (50 (link)). Briefly, the embryos were fixed in 4% paraformaldehyde at 4 °C overnight. After dehydration and hydration in a graded series of methanol and PBS + 0.1% Tween, the embryos were treated with Protease III (#322102) for 20 min. The following C1, C2, and C3 probes were used for hybridization at 40 °C overnight: LacZ (#313451-C1), Nkx2-5 (#428241-C2), Tbx5 (#519581-C2), Tnnt2 (#418681-C3), Hand1 (#429651-C3), and Mab21l2 (#456901-C3). Following this, 0.2 × saline sodium citrate buffer (SSC) + 0.01% Tween was used for washing in between steps. The embryos were incubated with AMP1 for 30 min, AMP2 for 30 min, and AMP3 for 15 min at 40 °C. For signal detection, the embryos were incubated in HRP-C1, -C2, or -C3 for 15 min at 40 °C, followed by incubation in Opal 520, 690, and 570 (Akoya Biosciences #FP1487001KT, FP1497001KT, FP1488001KT). HRP was inactivated by HRP-Blocker for 15 min at 40 °C. After staining, the embryos were embedded in 50% glycerol/PBS. Fluorescent images were captured using an FV3000 confocal microscope (Olympus) with a Z-stack every 3 μm.