Dako omnis system

In the UKB cohort, we assessed immune cell infiltration and TIGIT protein expression on whole slides by immunohistochemistry (IHC). Briefly summarized, paraffin sections of 4 µm thickness were cut from the tissue block and subsequently stained using the Dako Omnis system (Dako/Agilent Technologies). For IHC, we used mouse monoclonal anti-TIGIT antibody (cat. no. DIA-TG1, Oncodianova GmbH GmbH, Hamburg, Germany; dilution 1:50). We performed heat-induced antigen retrieval with target retrieval solution at pH 6 for 10 min at 117 °C using a steam pressure cooker. The slides were incubated with the primary antibody overnight at 4 °C. Signal detection was performed with an Alkaline Phosphatase Red Detection Kit (Dako/Agilent Technologies, cat. no. K5005). The slides were finally counterstained with hematoxylin and bluing reagent, dehydrated, and mounted. Tonsillar tissue was used as positive control. TIGIT protein expression in the tumor was assessed using the H-score rating the percentage of tumor cells negative (0), weak (1), moderate (2), and strong (3) (H-score: 0–300). Immune cells were scored according to TCGA (lymphocyte score [39 (link)]) and TIGIT⁺ immune cells were assessed as percentage fraction from all cells (TIGIT⁺ lymphocyte score).

Routine-based histopathologic evaluation was performed on formalin-fixed and paraffin-embedded (FFPE) material from surgical specimens. Immunohistochemical staining was performed using an automated Dako OMNIS system (Dako-Agilent, Santa Clara, CA, USA). The ACE2 polyclonal antibody was from BIOSS Antibodies (code bs-1004R). Positive (small intestine) and negative (only secondary antibody) were included in the test (Supplementary Figure S1). Quantification of lymphocyte subpopulations was performed using the Aperio digital slide scanner and an in-house developed algorithm within ImageScope (Leica Microsystems, Nussloch, DE, Germany) as previously described [18 (link)].

TS and ZEB1 immunohistochemical stains were performed in 71 PM biopsies using an automated platform (BenchMark, Ventana Medical Systems, Basel, Switzerland). Briefly, samples were pretreated for 36 min with antigen retrieval ULTRA CC1 then they were incubated for 40 min at 36° with TS (Ab 108,995, Clone EPR4545, 1:150 dilution, Abcam, Cambridge, UK) and ZEB1 (Ab HPA027524, polyclonal, 1:100 dilution, Sigma-Aldrich, St. Louis) primary antibodies. Both antibody staining scores were assessed by a pathologist (L.R.) using a semiquantitative histological score (H-score) as previously described [35 (link)].
BAP-1 (Ab sc-28383, clone C-4, 1:400, Santa Cruz, CA, USA) and p53 (Ab IR61661-2, clone DO-7, Ready-to-Use, Agilent Technologies, CA, US) immunostainings were performed using Dako Omnis System (Agilent Technologies, TX, US). BAP1 and p53 result interpretation was performed according to literature data [36 (link), 37 (link)].
Silver impregnation stain kit (Diapath, Martinengo, Italy) was used for reticulin fibers detection according to the manufacturer’s protocol.