Leica refine detection kit

Immunostaining of sponge slices was performed using a Leica Staining Robot with robotic antigen retrieval for F4/80 using trypsin solution (0.5 mg/mL in PBS, 10 min, 37 °C), or for αSMA manual antibody retrieval using a pressure cooker under standard conditions (citrate NCL pH 6 buffer); antigen retrieval was not performed for CD31. For F4/80 and αSMA, samples were blocked (5 min) with hydrogen peroxide solution from the Leica Refine Detection Kit (Leica Biosystems, Milton Keynes, UK) followed by serum block (30 min; F4/80, Immpress anti-rat (mouse absorbed) kit (Vector Laboratories, Peterborough, UK); αSMA, Mouse on Mouse Abcam Kit (Cambridge, UK)). Thereafter, samples were incubated with primary antibody (30 min; F4/80 1:300 (eBiosciences, Hatfield, UK); αSMA 1:4000 (Sigma-Aldrich, Dorset, UK)) prior to incubation with polymer (30 min, F4/80 Impress Kit as above; αSMA as above). For CD31 a Leica Refine Kit (as above) was used, which included a hydrogen peroxide block (5 min), primary antibody (120 min, 1:200 (Abcam)) and polymer incubation (15 min). Immunostaining was completed with incubation with 3,3′-diaminobenzidine (10 min) and counterstaining with haematoxylin (5 min), both from the Leica Refine Kit.

Subcutaneous xenograft tumors were prepared for IHC analysis. IHC was performed in a Leica Bond Max automated system (Leica Biosystems, Nussloch, Germany) using the Leica-Refine detection kit (Leica Biosystems, DS9800). Briefly, after deparaffinization, rehydration, and antigen retrieval, the sections were incubated with an anti-Ki-67 antibody (1 : 200, Abcam) and an anti-NF-κB p65 antibody (1 : 100, Abcam) for 30 min at room temperature. After washing, the sections were incubated with a horseradish peroxidase-conjugated secondary antibody, visualized with diaminobenzidine, and counterstained with hematoxylin. The stained slides were observed under a microscope (Olympus, Japan).

Protein lysates were subjected to electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Blots were incubated using primary anti-PD-L1, JAK2, pJAK2 (Y1007/1008), STAT1, pSTAT1 (Y701) (Cell Signaling Technology, Beverly, MA, USA), IRF-1, EBNA1, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Blots were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and visualized using ECL (Amersham Biosciences, Buckinghamshire, UK). Immunohistochemical analysis was performed using the Leica BOND-MAX autostainer and Leica Refine detection kit (Leica Biosystems, Melbourne, Australia) using primary PD-L1 antibody (1:50, clone E-7, Santa Cruz Biotechnology).

Immunohistochemistry was performed using a Leica Bond-Max (Leica Biosystems, Bannockburn, IL, USA) with a Leica Refine detection kit (Leica Biosystems). Antibodies and conditions for immunohistochemistry are shown in Supplementary Table S7.

Immunohistochemistry was performed using the Leica Bond-Max automation and Leica Refine detection kit (Leica Biosystems, Bannockburn, IL). The conditions for immunostaining have been summarized in Table 5. Signals were visualized by DAB. Mesothelin immunoreactivity (luminal/membranous or cytoplasmic) was evaluated with a detection cut-off of 5%. To compare the mesothelin positivity in small rectangular tissue samples on the tissue array and the corresponding larger whole-slide donor samples, 31 cases were selected for comparative immunohistochemical analysis. The results showing a high concordance are illustrated in Supplementary Figure 2. Immunohistochemistry of MLH1, MSH2, MSH6, and PMS2 were performed as previously reported [26 (link), 27 ].