Envision system reagents

In human pancreatic cancer tissue, IHC staining was carried out using Zt/f2 (5 μg/mL) as the primary antibody for RON and rabbit anti-MET mAb (1:100, 51,067, abcam) for MET, followed by EnVision System reagents (Dako, Carpentaria, CA, USA) as previously described (35 (link)). The negative control was performed by replacing the primary antibody with isotype-matched mouse IgG (5 μg/mL) (Figure S3). Human normal/benign pancreas tissue were used as the negative control. Two pathologists without knowledge of the patients' clinical records examined and scored the sections. Five tumor fields under ×400 magnification were randomly selected. Cytoplasmic and/or tumor cell membrane staining were considered to indicate positive expression. RON and MET expression were determined using a semiquantitative system as previously described (35 (link)). The proportion of positive cells was scored as follows: 0 (<5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The staining level was evaluated as follows: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown). The sum score, determined by adding up the positive proportion score and the staining level score, was as follows: 0 (negative; 0+), 1–3 (weakly positive; 1+), 4–5 (moderately positive; 2+), and 6–7 (strongly positive; 3+).

The amount of virus was evaluated using quantitative PCR. Organ tissue was prepared for haematoxylin eosin (HE) staining. Immunohistochemical (IHC) assays were also conducted. Paraffin sections of organs were de-waxed and then subjected to heat treatment in citrate buffer, and endogenous peroxidase activity was quenched using 0.3% H₂O₂ in methanol. Sections were blocked for 1 h with 3% bovine serum albumin (BSA; Cat#H1130, Solarbio, Tongzhou, Beijing, China) in PBS and incubated sequentially overnight at 4°C with 1:200 dilution of polyclonal rabbit anti-H7N9 antibodies(Cat#GTX125989, GeneTex, Irvine, CA, USA). Antibody binding was detected using EnVision System reagents (Cat#K5007, DAKO, Glostrup, Denmark). All slides were counterstained with haematoxylin.

Haematoxylin eosin (HE) staining of organ tissues was performed. The organ tissues were also subjected to Immunohistochemistry (IHC) staining. Organ tissues were embedded in paraffin and sectioned. The sections were then de-waxed and heated in citrate buffer. H₂O₂ (0.3%) in methanol was used to quench endogenous peroxidase activity. Sections were blocked for 2 h with Three percent bovine serum albumin (BSA; Cat#H1130, Solarbio, Tongzhou, Beijing, China) in PBS was used to block the sections for 2 h. The sections were then incubated at 4 °C overnight (12 h) with a 1:200 dilution of rabbit polyclonal antibodies recognizing H7N9 (Cat#GTX125989, GeneTex, Irvine, CA, USA). EnVision System reagents (Cat#K5007, DAKO, Glostrup, Denmark) were used to detect the bound antibodies. HE was used to counterstain all the slides.