Tissues were fixed in 4% paraformaldehyde, embedded in paraffin wax, and sectioned at 5 µm intervals. Slides were stained with hematoxylin using standard protocols, as previously described (Herriges et al., 2014 (link); Swarr et al., 2019 (link)). Immunofluorescence staining was performed using the following antibodies: anti-NKX2.1 (guinea pig, Seven Hills, 1:500), anti-SOX9 (rabbit, Millipore, 1:100), anti-KI67 (mouse, BD Pharmigen, 1:100), anti-SCGB1A1 (rabbit, Seven Hills, 1:500), anti-TUB1A1 (mouse, Sigma Aldrich, 1:1000), and anti-SOX2 (mouse, Santa Cruz, 1:100). Immunofluorescence for phosphorylated-ATK (pAKT) was performed with anti-pAKT (rabbit, Cell Signaling, 1:100), followed by multi-step detection with the Biotin-Tyramide signal amplification kit (Perkin Elmer) with a 15-min exposure time. TUNEL staining was performed using the TACS-XL Blue Label kit, per manufacturer’s instructions (Trevigen). All slides were mounted with Prolong Gold Antifade medium (Invitrogen), and were then imaged on either a Nikon Eclipse 90i widefield, or Nikon A1R GaAsP Inverted Confocal Microscope. Cell type quanitification based on immunofluorescence microscopy images was performed using the Nikon Elements Advanced Analysis software suite.
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