Immunostaining

Lung lobes were fixed overnight in 10% formalin in PBS before paraffin embedding. Paraffin sections were deparaffinized with Histo-Clear (National Diagnostics HS-200) and gradually rehydrated from ethanol to water. Antigen retrieval was carried out in citrate-based unmasking solution (Vector Laboratories H-3300) by microwaving at full power until boiling, then 30% power for 25 min (DLL3) or 15 min (HES1), then left to cool at room temperature for 10 min before washing with water. Endogenous peroxidase was blocked by incubating slides in 3% hydrogen peroxide for 1 h. Sections were washed in PBST (PBS with 0.1% Tween20), blocked in 5% horse serum for 1 h, and incubated with anti-DLL3 (1:200 Invitrogen PA5-23448) or anti-HES1 (1:200 Cell Signaling Technology 11988) diluted in PBST overnight at 4°C. DLL3 was developed using ImmPRESS®HRP Horse Anti-Rabbit IgG Polymer Detection Kit (Vector Laboratories MP-7401) following the manufacturer’s protocol. HES1 was developed using ImmPRESS® Excel Amplified Polymer Staining Anti-Rabbit IgG Peroxidase Kit (Vector Laboratories MP-7601) following the manufacturer’s protocol. All sections used DAB substrate kit (Vector Laboratories SK-4100) for color development. Sections were counterstained with hematoxylin (Sigma-Aldrich HHS32-1L), gradually dehydrated from water to ethanol to xylene, and mounted with Refrax mounting medium (Anatech Ltd711). Sections were imaged using Keyence BZ-X700 all-in-one fluorescence microscope with BZ-X Viewer program version 1.3.1.1 and BZ-X Analyzer 1.4.0.1.