Biotin xx tyramide superboost kit

The Biotin-XX-Tyramide Superboost kit (Invitrogen, USA, #2005939) was used for the TSA reaction. The rabbit anti lamin-B1 antibody (Abcam, USA, #ab16048) and a Streptavidin conjugate to Alexa 488 (1:200, Biolegend, #405235) were used for fluorescence staining.

Slides were deparaffinized and steamed for 45 min in Target Retrieval Solution (Dako Cat. S169984-2). Primary antibodies and dilutions used were as follows: TROP2 (Abcam, ab214488, 1:200), CEACAM5 (Agilent, M7072, 1:20), and PSMA (Agilent, M3620, 1:20). PV Poly-HRP Anti-Mouse IgG (Leica Microsystems Cat. PV6114) or Anti-Rabbit IgG (Leica Microsystems Cat. PV6119) was used as secondary antibody. Further signal amplification was done for CEACAM5 immunostains by using the Biotin XX Tyramide SuperBoost kit (Life Tech Cat. B40931). DLL3 staining was carried out on a Roche Benchmark Ultra instrument (Roche) using DLL3 (Ventana, SP347, 790-7016, 1μg/ml) and the CC1 module. DAB was used as the chromogen and counterstaining was done with hematoxylin and slides were digitized on a Ventana DP 200 Slide Scanner (Roche). Immunoreactivity was scored in a blinded manner by two pathologists (M. P. R., E. S.), whereby the staining level (“0” for no brown color, “1” for faint and fine brown chromogen deposition, and “2” for prominent chromogen deposition) was multiplied by the percentage of cells at each staining level, resulting in a total H-score with a range of 0–200. Note that PSMA and CEACAM5 expression data re-analyzed in this study were published previously by our group²⁰ .