Haematoxylin 2

The label was performed on Automate Discovery XT (Roche Ventana). In all, 5-µm sections of tissue were deparaffinized with Néo Clear (Dako) and two baths of absolute ethanol. Antigenic sites were unmasked with cell conditioning 1 (CC1) pH 8 (Roche). Sections were incubated with an anti-α-SMA antibody (Abcam) 1 h at 37 °C and with an OmniMap anti-rabbit HRP (Multimer; Roche) 16 min for the detection. Sections were incubated in Haematoxylin II (Roche) 16 min and with Bluing reagent (Roche) 4 min. Sections were dehydrated in two baths of absolute ethanol 1 min and xylene substitute Néo Clear (Dako) and mounted in Pertex (Histolab). Sections were scanned and labelling was quantified with CaloPix software.

Formalin-fixed and paraffin-embedded (FFPE) archival tumour blocks were analysed by immunohistochemistry by collecting 4 μm sections on Superfrost charged slides (ThermoScientific). After drying overnight at 37 °C, samples were processed using a Ventana Benchmark immunohistochemistry platform (Roche) with antibodies against p53 (Agilent cat#7001, RRID: AB_2206626, 1:50), CK7 (Agilent cat#7018, RRID: AB_2134589, 1:250), PAX8 (Roche cat#760–4618, 1:100). Heat induced epitope retrieval was performed using CC1 (Roche), incubating samples at 95 °C for 36, 52, and 40 min for p53, CK7 and PAX8, respectively. Antibodies were incubated at 37 °C for 32, 40 and 32 min for p53, CK7 and PAX8, respectively. p53 and CK7 were detected using Ultraview universal DAB kit (Roche), while PAX8 was detected using Optiview universal DAB kit (Roche), all as per manufacturer’s instructions. Sections were counterstained using Haematoxylin II (Roche) for 12 min and bluing reagent (Roche) for 8 min. Slides were imaged using the EVOS FL Auto 2 Imaging System (Invitrogen), using a × 10 or × 40 objective lens under bright field, and processed using Adobe Photoshop.

Automated immunostaining was performed on the Ventana Ultra (Roche). The protocol involved dewaxing with Ezprep solution, followed by heat-induced epitope retrieval with CC1buffer for 64 min and then endogenous peroxidase inhibition. Slides were then incubated with the ready to use BRAF V600E mutation-specific monoclonal antibody, (Ventana, clone VE1 (CE-IVD)) for 16 min at 37 °C. Chromogenic detection was carried out using the OptiView DAB IHC kit (Roche) along with an Optiview amplification kit (Roche). Four minutes Optivew amplification H2O2 and four minutes Optiview amplification multimer incubation times were used. Slides were counterstained with haematoxylin II (Roche) for 4 min followed by bluing reagent (Roche) for 4 min.
Positive staining was seen as the presence of unequivocal cytoplasmic granular staining of any intensity in the tumour cells. Negative staining showed the absence of cytoplasmic staining in the tumour cells.