A1r si point scanning confocal

TgBAC (gata5: EGFP) pd25 embryos at the right developmental stages were fixed in 4% paraformaldehyde at 4 ℃ overnight. Prehybridization, hybridization and the saline-sodium citrate (SSC) washes were performed at 65°C 85 . Detection of Digoxygenin (DIG) labeled probes were performed using an anti-DIG antibody, conjugated with horse-radish peroxidase (POD) (Roche Cat#11207733910) 1: 5000 followed by incubation with 1:50 to 1: 100 TSA plus Cyanine 3 solution (Perkin Elmer). To detect two genes at a time, embryos were incubated with a probe mix containing DIG and Flu-labelled probes. Fluorescein labeled probes were detected using an anti-Flu-POD antibody (Roche Cat#11426346910) and deposition of 1:50 to 1: 100 TSA plus Cyanine 3 solution. DIG-labelled probes were detected using an anti-Dig-AP antibody, developed with FastBlue (Sigma, F3378) and NAMP (Sigma, N5000), and monitored under a dissecting scope. GFP was detected using primary antibody Rabbit anti-GFP 1:500 (Torrey Pines Biolabs) and secondary antibody 488 Goat anti Rabbit (Thermo Scientific, Cat# A-11008) 1:1000. Embryos were stained for DAPI 1:2000 before mounting in low melt agarose for imaging under a Nikon A1R Si Point Scanning Confocal at 20X and 40X magnification.

Bright-field images and were taken using a Zeiss AXIO Zoom V16. Confocal microscopy was performed under a Nikon A1R Si Point Scanning Confocal at 40X magnification to determine the tcf21+ cell number in TgBAC(tcf21: NLS-EGFP) pd41 embryos and to image heart morphology in Tg(myl7:EGFP) twu34 embryos. The numbers of tcf21+ cells were counted using the Cell Counter plugin in Fiji (https://imagej.nih.gov/ij/index.html).