Fluorescent Whole-Mount In Situ Hybridization

For fluorescent whole mount in situ hybridization (FISH), we followed the protocol outlined in (69 (link)). Triple FISH was performed as described in (70 (link)). Signal was developed with fluorophore-conjugated tyramide (1:400 reagent diluents, Perkin Elmer). Labeled probes were transcribed from linearized DNA using digoxigenin-11-UTP, fluorescein-12-UTP (Roche, Indianapolis, IN, USA), or labeled with DNP (Mirus, Madison, WI, USA) following kit instructions. SpLox, SpBrn1/2/4, SpSoxC, SpPtf1a, and SpMist probes were made as previously published [SpLox (71 (link)), SpBrn1/2/4 (25 (link)), SpSoxC (69 (link)) SpPtf1a, and SpMist (53 (link))]. SpIsl, SpNgn and SpNeuroD probes were synthetized using the following primers: SpIsl-F: 5′-CGTGGACCAGACAGACTTGA-3′; SpIsl-R: 5′-AGTCGCTGAGTGCTTTCCAT-3′; SpNgn-F: 5′-TACGACAATGATGCCCAAGA-3′; SpNgn-R: 5′-CCGTTTCACAAAGCCATTTT-3′; SpNeuroD-F: 5′-CTCGCCACCTGATCTCTAC-3′; SpNeuroD-R: 5′-TTCCCGCCTTTCAAAATATG-3′. SpANP2 probe was made as published in Woods et al. 2018. Templates of all the probes were sequenced prior to probe generation and cloned in the pGEM®-T Easy Vector (Promega, Madison, WI, USA). Samples were imaged with a Zeiss 510 Meta confocal microscope.