Whole-Mount In Situ Hybridization for Embryonic Gene Expression

Embryos were fixed in 4% paraformaldehyde at 4°C and gradually dehydrated in methanol. Whole-mount in situ hybridizations were performed using DIG-labeled anti-sense RNA probes (made with labeling kit: Roche, 11175033910, Basel, Switzerland) to sizzled [76 (link)], foxi1 [37 (link)], and bambia. Probes were visualized with anti-DIG-Horseradish Peroxidase (Roche, 11207733910, Basel, Switzerland) developed with TSA Plus Cyanine 3 kits using a 1:50 dilution (Perkin Elmer, NEL744001KT, Waltham, Massachusetts, USA), and nuclei were stained with 1:1,000 dilution of Sytox Green. Embryos were cleared, mounted, and imaged as described for immunofluorescence. Images were analyzed using the same MATLAB algorithm, except fluorescent intensity was extracted from a 25-pixel sphere from the center point of each nucleus to include the cytoplasmic staining.