Ventana discover ultra

Briefly, 4 μm paraffin mouse lung tissue sections were mounted on glass slides and were deparaffinized. The immunostaining was performed on the Ventana Discover ULTRA autostainer (Ventana Medical Systems, Oro Valley, AZ) using OmniMapHRP detection method. The primary antibody was anxA1 antibody (clone 4-huIgG1) and was incubated with concentration at 0.5 μg/mL Following primary antibody incubation, the samples were incubated in the specific link antibody rabbit anti-human IgG at concentration 2 μg/ml (Jackson ImmunoResearch Laboratories® cat# 309-005-082) for 16 minutes. Then, primary antibody was visualized with OmniMap goat anti-rabbit HRP (catalog no. Cat #760–4311, Ventana) respectively, and DAB (catalog no. cat# 760–159, Ventana).
All IHC stained slides were assessed by an experienced board-certified pathologist (JAC). The intensity of Annexin A1 IHC staining was assessed semi-quantitatively: 0 (none), 1+ (faint) 2+ (moderate), 3+ (maximum); and the extent of staining was estimated as the percentage of tissue stained positively. In the tumor samples, neoplastic cell staining was assessed as present or absent.

Tumour tissue and blood samples were collected from all study participants for biomarker analysis, including CD73 expression. Adequate tumour tissue was defined as a formalin-fixed and paraffin-embedded (FFPE) tumour block or approximately 10 unstained slides, each 4–5 µm thick. An archival FFPE sample taken within 3 years prior to screening was preferred. Immunohistochemistry (IHC) of tissue biopsy specimens for CD73 and programmed death-ligand 1 (PD-L1) was performed. CD73 IHC was performed with the anti-CD73 antibody clone [EPR6115] (ab124725) and the Ventana Discover ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA). PD-L1 IHC was performed with the anti-PDL1 antibody clone (SP263) and the Ventana Benchmark ULTRA autostainer. Following immunostaining, slides were scanned using an Aperio AT turbo or an Aperio XT scanner with a 20 × microscope objective. For blood samples, approximately 3.5 mL of whole blood was collected at each time point, from which > 0.5 mL of serum sample was obtained. The methods for ligand binding analysis of soluble CD73 are summarised in Online Resource 1.