Ascl1 clone 24b72d11

IHC and IF studies using ASCL1 (clone 24B72D11.1, catalog number 556604, BD Biosciences, San Jose, CA) and NEUROD1 (clone EPR17084, catalog number ab205300, Abcam, Cambridge, MA) specific antibodies were carried out on archival formalin-fixed paraffin-embedded tissues. In brief, 5 μm paraffin sections were de-waxed and rehydrated following standard protocols. Antigen retrieval consisted of steaming for 40 min in Target Retrieval Solution (S1700, Agilent, Santa Clara, CA). Slides were then washed and equilibrated in TBS-Tween buffer (Sigma, St. Louis, MO) for 10 min. Primary antibodies were applied at a dilution of 1:25 at 37 °C for 60 min. For chromogenic studies, immunocomplexes were visualized by applying secondary detection reagents of the UltraVision™ Quanto Detection System (catalog number TL-060-QHD, Thermo Fisher, Waltham, MA) following the manufacturer’s instructions. Sequential dual-IF labeling studies were carried out using Tyramide SuperBoost kits (Thermo Fisher, Waltham, MA). All bright-field slides were imaged using a Ventana DP200 system (Roche Diagnostics, Indianapolis, IN). Fluorescence images were acquired on a Cytation 5 Cell Imager (Biotek, Winooski, VT). All the slides have been evaluated by an expert pathologist and the stainings have been replicated a minimum of three times.

TMAs and slides were stained for subtype‐defined markers of SCLC including ASCL1 (clone 24B72D11.1, dilution 1:100; BD Biosciences), NEUROD1 (clone EPR17084, dilution 1:50; Abcam), POU2F3 (clone 6D1, dilution 1:100; Santa Cruz), YAP1 (clone 63.7, dilution 1:2000; Santa Cruz) and Vimentin (clone D21H3, dilution 1:100; Cell Signaling Technologies) using Anti‐mouse/rabbit IHC Detection Kit (PK10006; Proteintech) according the manufacturer's protocol. Two independent pathologists reviewed the stained slides in a blinded fashion. The expression of each marker from tumor cells was assessed by histoscore (H‐score, range 0–300), which was calculated by multiplying the proportion of positive tumor cells (0%–100%) by the intensity of positive staining (no staining = 0, weak staining = 1, moderate staining = 2, and strong staining = 3).³⁷, ³⁸ A dominant marker is defined as the marker with the highest H‐score. In SCLC with combined SCLC and NSCLC components, IHC scores reflect expression exclusively in the SCLC component.