Axiovision m1

Urogenital tissues were dissected from chicken embryos and briefly fixed for 15 min in 4 % paraformaldehyde/PBS at room temperature. Tissues were then cryoprotected by immersion in 30 % sucrose/PBS (overnight at 4 °C), infiltrated with OCT embedding compound. Ten micron frozen sections on slides were treated as previously described [63 (link)]. Secondary antibodies were AlexFluor 488 donkey anti-rabbit IgG, 1:1000, and AlexaFluor 594 donkey anti-mouse IgG, at 1:1500 from Invitrogen). Sections were mounted in Fluorosave (Calbiochem). Images were taken on a Zeiss Axiovision M1.

The CARD-FISH procedure was performed with Alexa 488-labeled tyramides (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA), as previously described (56 (link)), and analyzed manually using 10 to 20 microphotographs randomly taken by epifluorescence microscopy at ×1,000 magnification (AxioVision.M1; Carl Zeiss, Jena, Germany). Biovolume was estimated by multiplying cell abundance by average cell volume, which was calculated based on manual measurements of cell width and length and assuming the cell shape to be prolate spheroid, as described by Piwosz in 2019 (46 (link)). The relative abundance of an individual lineage was calculated as the proportion of cells hybridized with the specific probe to that of cells hybridized with the general eukaryotic probe. A full list of applied probes (n = 11) can be found in Table S2.