Dako blocking reagent

Immunohistochemistry was performed to assess FoxO1 protein expression in foetal thymus, lung and liver tissues. Five‐micrometre‐thick sections of FFPE blocks of foetal thymus, lung and liver tissues from autopsy cases were placed on silanized slides. For antigen retrieval, the sections were heated in citrate buffer (pH 6.0) for 30 min. using a microwave. Then, endogenous peroxidases were quenched with DAKO blocking reagent (Dako Corp., Carpinteria, CA, USA) for 15 min. The sections were incubated with a rabbit monoclonal anti‐FOXO1 antibody (1:100, Cell Signaling) and subsequently incubated with a biotinylated anti‐immunoglobulin and streptavidin‐peroxidase (DAKO LSAB kit, Dako Corp.). Rabbit mAb IgG isotype (1:100; Cell Signaling) was used as a negative control. Colour was developed using 3, 3‐diaminobenzidine tetrahydrochloride (DAB) for 3 min., and then, the sections were counterstained with haematoxylin for 1 min.

For IHC, paraffin-embedded sections (3.5 μm) were dewaxed and rehydrated. Antigen retrieval was performed by incubation in sodium citrate buffer pH 6.0 at 65 °C overnight. After cooling, unspecific antigens were blocked with the DAKO blocking reagent (Dako, Glostrup, Denmark), followed by overnight incubation with the 1:250 diluted rabbit monoclonal antibody [EPR9514] against human p-MLKL (phospho S358; abcam, Cambridge, UK), human cleaved caspase 8 (NB100-56116; NOVUS Biologicals, Centennial, CO, USA) or RIPK3 (GTX107574; GeneTex, Irvine, CA, USA) at 4 °C. Sections were treated with 3% hydrogen peroxide before starting the staining with the Dako LSAB2 System-HRP kit (Dako, Glostrup, Denmark). In all samples a final staining of cell nuclei by Gill’s hematoxylin No 3 (Sigma-Aldrich) was performed. In case of cleaved CASP8 and p-MLKL, the percentage of positive cells was quantified by manual counting of the stained sections.

Upon sacrifice, lungs were inflated with a mixture of 1:1.5 OCT:PBS using a 26G needle. Inflated lungs were then embedded in OCT or FSC 22 Clear Frozen Section compound (cat. no. 3801480, Leica) and snap frozen and stored at −80 °C. Lungs were cryo-sectioned at 12 µm and then stained for expression of Ki67. Sections were allowed to thaw for 10 min, and then fixed in ice cold acetone (5 min). Endogenous peroxidases were quenched with 1% aqueous H₂O₂ in methanol for 15 min and then blocked in Dako Blocking reagent (cat. no. X0909, Dako) supplemented with 10% donkey serum (no. 017-000-121, Jackson ImmunoResearch) for 30 min. Ki67 (D3B5) Rabbit mAb (Mouse Preferred; IHC Formulated) (cat. no. 12202, Cell Signalling Technologies) was incubated overnight at 4 °C at a dilution of 1:200 in Dako antibody diluent with background reducing components (cat. no. S3022, Dako). After 3 washes in PBST, sections were incubated with LSAB2 Streptavidin-Peroxidase/SA-HRP kit (DAKO K0690) for 30 min, and then mounted using Permount (cat. no. # SP15-500, Fisher Scientific). Images were acquired on a Leica DM LB2 microscope and DFC 300 FX camera. ImageJ was used to determine the pixel area of the lung and also pixel area of metastatic nodules, from which the percentage metastasis area was calculated.

The immunohistochemistry (IHC) for EphA2 was performed manually according to standard procedure, on paraffin-embedded PDX tumor tissues. The antigen retrieval was performed with 15-min sub-boiling citrate buffer (pH = 6). Next, a peroxidase blockade was carried out with serum-free Dako blocking reagent (Agilent Technologies, Inc., Santa Clara, CA, USA). The primary antibody for EphA2 staining was D4A2 XP^® Rabbit mAb #6997 (Cell Signaling Technologies, Danvers, MA, USA) diluted 1:200 and incubated overnight at 4 °C. Staining was obtained with Dako EnVision+ System- HRP Labelled Polymer Anti-Rabbit (Agilent Technologies, Inc., Santa Clara, CA, USA) followed by 3,3′-Diaminobenzidine as the standard chromogen and counter-staining with hematoxylin. Slide fixation was performed with mounting medium and observation under an optical microscope (Leica DM750, Leica Biosystems, Buccinasco, MI, Italy) equipped with a Leica ICC50W camera (Leica Biosystems).