Envision flex

IHC was performed using an Autostainer Link 48 (Dako). The deparaffinized sections were exposed to EnVision FLEX target retrieval solution high pH (Agilent, Cat# K8004) for 20 min at 97 °C for activation, and mouse anti-SARS-CoV-2 N monoclonal antibody (R&D Systems, Clone 1035111, Cat# MAB10474-SP, 1:400) was used as a primary antibody. The sections were sensitized using EnVision FLEX (Agilent) for 15 min and visualized by peroxidase-based enzymatic reaction with 3,3′-diaminobenzidine tetrahydrochloride as a substrate for 5 min.
For the evaluation of N protein positivity in the tracheae at 2 d.p.i. and the lung specimens of infected hamsters at 2 and 5 d.p.i. (B1.1, BA.1, BA.2, and BA.5, n = 4 each), staining was performed with mouse anti-SARS-CoV-2 N monoclonal antibody (1:400). N-protein positivity was evaluated by certified pathologists as previously described^{9 (link),11 (link)}. Images were incorporated as virtual slides by NDP.scan software v3.2.4 (Hamamatsu Photonics). N-protein positivity was measured as the area using Fiji software v2.2.0 (ImageJ).

Sections were deparaffinized and rehydrated as described above. Antigen retrieval was performed at 110°C for 5 minutes in Target Retrieval Solution (pH 9.0; Dako, #S2367) in a RHS-2 microwave processor (Milestone, Sorisole, Italy) within a pressurized reaction vessel. Endogenous peroxidase activity was quenched by incubating the sections in 3% hydrogen peroxide for 20 minutes at room temperature and non-specific binding was blocked by incubating in serum-free protein block (Dako, #X0909) for 20 minutes at room temperature. Sections were labeled with a rabbit anti-RET monoclonal antibody (1:1000 dilution, clone EPR2871, Epitomics, Burlingame, CA, USA; see Additional file 1 for antibody specificity and immunohistochemistry validation; Additional file 1: Table S1) and RET expression was visualized with EnVision™ FLEX+ (Dako, #K8012). Sections were counterstained with hematoxylin. Cell lines (TT, MiaPaCa, SKNMC and Panc1) and tissue samples (human non-inflamed appendix and in-house NSCLC tissue microarray) were used as controls for RET immunostaining.

Immunohistochemistry was performed on FFPE tissue sections using the Dako Autostainer link platform. Deparaffinization, rehydration, and target retrieval were performed in the PT Link (Dako PT100). Slides were then processed on the Autostainer Link 48 (Dako AS480) using an automated EnVision FLEX (DAKO) staining protocol. The anti‐ERRFI1 mAb clone E2 was used at 5 μg/ml (Frosi et al, 2010). Positive and negative controls were included for each immunohistochemical run. Pictures were acquired with the Leica Assistant Suit (LAS EZ) Software.

According to Dako's recommendations, sections were stained with CD10 immuno-stain using labeled and enhanced polymer systems (Dako EnVision™ Flex). In the Dako autostainer PT link, staining steps and incubation times are pre-programmed. The sections with 4 µm thick were mounted on salinized slides and put in an autostainer (in which a substrate buffer, blocking reagent for endogenous peroxidase, monoclonal anti-human CD10 as a primary antibody, mouse linker as a secondary reagent, EnVision/HRP as a labeled polymer, chromogen, a counterstain as hematoxylin, and distilled water were applied on all slides). After removing the autostainer slides, they were put in graded ethanols of 70%, 100%, and 100% for 2 minutes each; then, they were put for 2 minutes in xylene. Lastly, mounting was performed using Canada Balsam. Scoring of stromal CD10 immunoreactivity was done and reviewed by two expert pathologists using light microscopy. A section from the reactive lymph node was used as a positive control for CD10 immunoreactivity, while the negative control was checked by omitting the primary antibody.

TRKA protein expression was assessed in formalin-fixed paraffin-embedded (FFPE) tumor tissue sections using a rabbit monoclonal antibody (clone EP1058Y; Epitomics, Burlingame, CA) that recognizes the carboxyl-terminus of the protein. Briefly, three micron-thick sections were reacted for 30 min with the anti-TRKA antibody used at 1:200 working dilution and then incubated with a commercially available detection kit (EnVision™ FLEX+, Dako, Glostrup, Denmark) following the manufacturer's instructions and previously refined IHC methods. The specificity of all reactions was checked replacing the primary antibody with a non-related mouse immunoglobulin at comparable dilutions or using normal serum alone, Positive and negative controls were used as appropriate KM12 cell line, known to carry a TPM3-NTRK1 gene rearrangement, was used as positive control.

Four micrometre thick sections were dewaxed and rehydrated through xylene and graded alcohol, respectively, quenched for endogenous peroxidase with 3% hydrogen peroxide in methanol (VWR International) for 15 min (minutes) at room temperature (RT), before epitope unmasking using pH6 (10 mM citric acid buffer, adjusted to pH6 with 1 M sodium hydroxide; Fisherscientific), pH9 target retrieval buffer (Envision FLEX, high pH; Dako) for 10 min at 97 °C, or proteinase XXIV (Sigma-Aldrich) for 15 min at RT. Slides were blocked with normal goat serum for 30 min in RT (1/66 dilution; Vector Laboratories) and assembled into cover plates to facilitate IHC using the Shandon Sequenza system (Shandon). Samples were then incubated with a primary antibody at 4 °C overnight or 1 h at RT, followed by incubation with mouse- or rabbit-specific Envision™ polymer (Dako) for 30 min at RT and visualised using 3,3-diaminobenzidine (DAB) (Sigma Aldrich) for 10 min at RT. Tris-buffered saline with Tween (145 mM NaCl, 5 mM Tris(hydroxymethyl)methylamine, 0.1% w/v Tween®20, adjusted to pH7.6 with 1 M HCl; Fisher Scientific; VWR International) were used for rinsing sections between incubations. Subsequently, sections were counterstained in Mayer’s haematoxylin (Surgipath), dehydrated in ethanol and xylene, and glass coverslips mounted using Dibutyl Phthalate Xylene.