Immunofluorescence Imaging and In Situ Hybridization

Cryostat sections were mounted in Vectashield (Vector Laboratories) or in 5 mg/mL propyl gallate in glycerol/PBS (9:1) with 0.01% azide. Whole mount explants were gradually dehydrated in methanol and cleared in methyl salicylate (Sigma) as described previously [31 (link),37 (link)]. Immunofluorescence images were taken on a confocal Leica SPE microscope, following imaging acquisition steps described previously [37 (link)]. Image analysis was performed using Fiji v. 1.49 (https://imagej.net/Fiji, accessed on 15 June 2022) software. Image histogram corrections and, when appropriate, maximum intensity projections of immunofluorescence confocal stacks were produced in Fiji. When applicable, contiguous images were stitched together into a single image using the pairwise stitching Fiji plugin [61 (link)]. Image acquisition of embryos and explants processed for in situ hybridization was performed using a Zeiss LUMAR V12 Stereoscope coupled to a Zeiss Axiocam 503 colour 3MP camera. Imaging of explants prior to in situ hybridization was performed in 50% formamide solution. For comparing the in situ hybridization patterns along the paraxial mesoderm, the Fiji plugin Straighten [62 (link)] was used and contralateral explant pairs were aligned by SIV.