Nbp1 83966

FFPE Sections (4-μm thick) were mounted and routinely stained with H&E for histopathological examination (Supplementary Fig. S1a). Multiplex immunofluorescence was applied to identify the expression patterns of key transcriptomic regulators of SCLC, including ASCL1 (Abcam, ab211327), NEUROD1 (Abcam, ab60704) and POU2F3 (Novus Biologicals, NBP1–83966). Multiplex immunofluorescence staining was performed using a PANO 7-plex IHC kit (Panovue, Cat# 0004100100), as previously described.⁶⁵ In brief, the FFPE sections were subjected to deparaffinization, rehydration, and antigen retrieval according to the protocol supplied by the manufacturer. After blocking, the sections were incubated with a primary antibody and then a secondary antibody (polymer HRP-anti-mouse/Rabbit IgG). Other primary antibodies were sequentially applied by repeating the previous procedures. Nuclei were stained with DAPI (Sigma-Aldrich, D9542) after all the human antigens had been labeled. Multispectral images were obtained by scanning the stained slides with the Mantra System (PerkinElmer, Waltham, Massachusetts, US) and analyzed using inForm image analysis software (PerkinElmer, Waltham, Massachusetts, US) (Fig. 4c).

Consecutive four-micrometer-thick tissue sections were cut from FFPE tissues for IHC. IHC staining was performed using a Ventana automatic immunostainer (Ventana, Benchmark XT, Tucson, AZ, USA) following standard automated protocols. Ventana Retrieval Solution CCl (equivalent to EDTA buffer, pH 8.0) was used for epitope retrieval for 30 min. The primary antibodies (ASCL1:1:100, Clone EPR19840, Abcam, Cambridge, UK; NEUROD1:1:4000; Clone IMR-32, Abcam, Cambridge, UK; POU2F3:1:50, polyclonal, NBP1-83966, Novus Biologicals, Centennial, CO, USA) were incubated for 32 min at 42 °C and detected using the Ultra-View Detection Kit (Ventana) with DAB as the chromogen. FFPE cell line pellets with known protein expression of ASCL1, NEUROD1, and POU2F3 were used to establish optimal IHC conditions and assess the sensitivity and specificity of each antibody. The proportion of positive tumor cells was calculated as described previously [30 (link)].