Pt link pre treatment module

SARS-CoV-2 VIC01 infection of ferrets is described in Au et al. (2022 (link)). Briefly, ferret turbinates at 7 days post infection were fixed in 10% neutral buffered formalin (Australian Biostain). Preserved ferret turbinates were processed according to routine histological methods, embedded in paraffin wax (Leica Biosystems), and 4-µm serial sections were mounted on glass slides (Menzel Gläser). Tissue sections were deparaffinised using routine histological methods. Antigen retrieval was performed using Target Retrieval Solution, high pH (Dako) at 97°C for 30 min on a PT Link pre-treatment module (Agilent).

All samples were sectioned and stained on the same occasion for comparable analysis. Paraffin-embedded sections were placed in a PT Link Pre-treatment Module (Agilent) 97 °C for 20 minutes for deparaffinization, followed by incubation with Target Retrieval Solution, Citrate pH 6 (S236984–2, Agilent) for 30 minutes. Immunohistochemistry was performed in an Autostainer Link 48 (Agilent) using an EnVision FLEX visualization system (Agilent) and counterstained with hematoxylin-eosin. Tissue sections were incubated for 30 minutes at room temperature with primary antibody, a polyclonal rabbit anti-human VAP-1 antibody (1:100, PA5–81910 Thermo Fischer Scientific). Negative control sections were prepared by performing immunostaining procedures without adding primary antibodies. Stained sections were scanned by a digital slide scanner (NanoZoomer S60, Hamamatsu) using the same exposure times. Digitalized sections were examined by NDP.view2 (Hamamatsu), a whole slide viewing software. The same magnification (10 x objective) was used for all the images. Three representative areas per section/patient were exported into three images (size, 23 MP, 6400 × 3616 pixels, type: RGB, format: TIFF). RGB image allowed the range of 255 intensity levels in the three color channels (red, green, blue), no saturated pixels could be observed.

Freshly cut 4 µm tissue sections were stained for CD3⁺ T lymphocytes and the subset of CD8⁺ cytotoxic T-cells. Following deparaffinzation, slides were exposed to heat-induced antigen retrieval for at 98 °C in pH 9 target retrieval solution (Agilent, Santa Clara, CA, USA) in a PT Link pre-treatment module (Agilent) and stained in an Autostainer Link 48 device (Agilent) using primary antibodies against CD3 (Dako, rabbit polyclonal antibody, Santa Clara, CA, USA; #IR503; undiluted) and CD8 (DAKO, mouse monoclonal antibody, #IR623, undiluted). Protocol steps were performed according to the manufacturer′s directions and include 5 min peroxidase blocking (Agilent REAL) and 20 min of primary antibody incubation at room temperature followed by visualization of the bound antibody using the EnVision Flex Kit (Agilent).

For the immunohistochemical analysis, human normal and neoplastic tissues, including lung, breast, ovary, and stomach samples, were selected from the archives of the Department of Pathology, University of Palermo. Immunohistochemistry (IHC) was performed using a polymer detection method. Briefly, tissue samples were fixed in 10% v/v buffered formalin and then paraffin embedded. 4 µm-thick tissue sections were deparaffinized and rehydrated. The antigen unmasking technique was carried out using Novocastra Epitope Retrieval Solutions, pH 9 (Leica Biosystems) in a PT Link pre-treatment module (Dako) at 98°C for 30 min. Sections were then brought to room temperature and washed in PBS. After neutralization of the endogenous peroxidase with 3% v/v H₂O₂ and Fc blocking by a specific protein block (Novocastra, Leica Biosystems), samples were incubated overnight at 4°C with rabbit anti-human SP-D (dilution 1:300) polyclonal antibodies (MRC Immunochemistry Unit, Oxford, UK). Staining was revealed via polymer detection kit (Novocastra, Leica Biosystems) and AEC (3-amino-9-ethylcarbazole, Dako, Denmark) substrate-chromogen. Slides were counterstained with Harris Hematoxylin (Novocastra, Leica Biosystems). Sections were analyzed under the Axio Scope A1 optical microscope (Zeiss) and microphotographs were collected through the Axiocam 503 color digital camera (Zeiss) using the Zen2 software.

Expression of C-MYC was evaluated according to the guidelines for routine diagnostic work-up at the Department of Pathology, Herlev Hospital [11 (link)]. FFPE tissue sections were pre-treated (pH = 9.0) on a PT Link pre-treatment module (Dako), including paraffin removal, rehydration, and epitope retrieval, and subsequently stained in a Dako Autostainer Link 48, using EnVision FLEX+ visualization kits (Dako) and monoclonal C-MYC antibody Epitomics; clone y69/EP121, 1:100 dilution EnVision Flex Antibody Diluent). All steps were completed according to the manufacturer’s instructions. The stained sections (Fig. 1c) were evaluated and scored by two experienced haemato-pathologists using a double-headed microscope (Olympus BX51, equipped with a colour view camera and analySIS getIT 5.0 software (Soft Imaging Systems Munster, Germany)). MYC expression was evaluated on full slide sections in hot spot areas and all staining intensities were included as previously described [12 (link)].

Deparaffinization, rehydration, and antigen retrieval were performed using the PTlink pretreatment module (Dako, Les Ulis, France). Immunostaining was performed using a DAKO automaton or manually with the following primary antibodies: anti-CELF2, anti-Olig2, anti-MIB-1, anti-CCNA, anti-SOX3—as indicated in the Supplementary Information. Immunostaining was scored independently by a pathologist (F.B.V.) and a researcher (T.V.).