Discovery ultra stainer

Routine histochemical and immunohistochemical stains were used to characterize tumors. Tumors were fixed in 10% neutral-buffered formalin for 24 hours and processed to paraffin blocks. Serial histologic sections were stained for H&E or used in IHC assays, which were performed using a Discovery Ultra Stainer (Ventana Medical Systems) using reagents designed for Ventana instruments and antibodies directed against human DLL3 (Ventana SP347), CD4 or CD8 (Spring Biosciences or Cell Signaling Technology), and PD-1 or an isotype control antibody (Abcam). Antigen retrieval was performed on deparaffinized tissue using the BioSB TintoRetriever and either citrate or EDTA buffer based on the antibody manufacturer’s recommendations. Slides were incubated with the primary antibody or isotype control for 1 hour at room temperature and with OmniMap HRP secondary antibodies (Roche Diagnostics) for 15 minutes at room temperature. Complexes were detected using a Ventana DAB chromogen kit (Roche Diagnostics), and sections were counterstained with hematoxylin. Analysis of histologic sections was performed using a Nikon Eclipse 90i microscope, scanned using an Aperio scanner (Leica) and processed using ImageJ software. Archival FFPE human lymph node samples were used as a positive control for CD4, CD8 and PD-1 immunostaining.

Analyses were performed on 6μm formalin fixed, paraffin embedded (FFPE) sections and stained using an automated Ventana Discovery Ultra Stainer. Antigen retrieval using CC1 buffer (Roche, 950–500) was performed before incubation with primary antibodies, F4/80 (Cell Signaling Technologies, 70076S, concentration of 1:1000) and TMEM119 (Abcam, ab209063, concentration of 1:500). Antigens were detected using anti-rabbit-HQ (hapten) (Roche, 760–4815), anti-HQ-HRP (Roche, 760–4820), and ChromoMap DAB substrate (Roche, 760–159). A hematoxylin and bluing (Roche, 760–2021/760–2037) counterstain was applied to enhance visualization before the slides were dehydrated, cleared, and mounted.