Epitope retrieval solution

Tissue sections (4.0 μm thick) were dewaxed and rehydrated. Antigen retrieval was performed in an autoclave using Epitope Retrieval Solutions (Leica Biosystems, Newcastle, UK) at pH 9.0 for 10 minutes at 121.0°C. The slides were allowed to react with a c-MET specific antibody (clone SP44, 1:50 dilution; Ventana Medical Systems, Inc., Tucson, AZ, USA) at room temperature for 1 hour. The slides were incubated using an immunohistochemical stain detection kit (UltraVision^TM Quanto Detection System; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and counterstained with hematoxylin. For the negative controls, the primary antibody was replaced with 5.0% fetal bovine serum. Staining intensities were categorized as 0 (no staining), 1+ (weak cytoplasmic staining), 2+ (moderate cytoplasmic staining), or 3+ (strong cytoplasmic staining). MET positivity by immunohistochemistry was defined as a 2+ or 3+ staining intensity in ≥ 50.0% of the tumor cells according to the Metmab criteria used in the NCT01456325 study [14 (link)], a Metmab phase III trial of advanced non-small cell lung cancer patients.

Human tissue samples were collected according to the Helsinki Declaration and the study was approved by the University of Palermo Ethical Review Board (Approval number 05/2018 and 06/2021). Four-micrometers-thick sections were deparaffinized, rehydrated and unmasked using Novocastra Epitope Retrieval Solutions, either pH6 (Leica Biosystem, #RE7113-CE), or pH9 (Leica Biosystem, #RE7119-CE) in thermostatic bath at 98 °C for 30 min. After washing, slides underwent neutralization of the endogenous peroxidase with Novocastra Peroxidase Block (Leica Biosystems, #RE7101), and Fc bloking with Novocastra protein block (Leica Biosystems, #RE7102). Then, samples were incubated with primary antibodies (listed in Supplementary Material and Methods). After incubation with HRP-conjugated IgG (H&L) specific secondary antibodies (Life Technologies) or polymer detection kit (Novocastra, Leica Biosystems), signals were revealed with Novocastra DAB Chromogen (Leica Biosystems, #RE7105). Anti-mouse and anti-rabbit Alexa Fluor 488- and 568-conjugated secondary antibodies were used for fluorescence detection. Details on image acquisition and analysis are provided in Supplementary Material and Methods.

Immunohistochemical stainings were performed using 5 μm serial tissue sections of representative FFPE tumour samples. For SOX9 specific immunohistochemistry first a heat-induced epitope retrieval was done applying ProTaqs IV Anitgen-Enhancer (Quartett, Germany) before adding primary SOX9 specific rabbit monoclonal antibody (1:100, SOX9 clone D8G8H, Cell Signalling Technology, Danvers, MA) for 60 min at room temperature. Thereafter, a development step was introduced by adding detection-system (Vectastain ABC-Kit Elite Universal, Biozol, Germany) and subsequently substrate-chromogen containing DAB+ system (DAKO, Germany) according to the respective manufacturer’s protocols.
For SOX2 specific immunohistochemistry heat-induced epitope retrieval was performed applying Epitope Retrieval Solution (Novocastra, Germany) followed by adding primary SOX2 specific rabbit monoclonal antibody (1:50, SOX2 clone D6D9, Cell Signalling Technology, Danvers, MA) for 60 min at room temperature. The development step was made by adding the detection-system (ImmPRESS Reagent Kit Anti-RABBIT Ig, Vector, Germany) and substrate-chromogen containing DAB+ system (DAKO, Germany).
Finally, for both markers slides were counterstained using Hematoxylin (Vector Laboratories, Burlingame, CA). To control for unspecific binding of antibodies, isotype controls were included (data not shown).

For IF we used formalin-fixed paraffin-embedded 3-μm-thick tissue sections. After deparaffinization, the samples were buffered at 97°C with Epitope Retrieval Solution (Novocastra, Leica) at pH 6 for PDGFRα and pH 9 for CD34. The samples were washed in PBS with glycine (2 mg/ml) and then blocked with 2% BSA for 1 hr. Samples were incubated with primary antibodies overnight at room temperature with a cocktail consisting of mouse monoclonal CD34 (1:50, clone QBEnd10; Dako, Glostrup, Denmark) and rabbit polyclonal PDGFRα (1:40; Neomarkers, Fremont, CA, USA). After washing three times in EnVisionTM Flex Wash Buffer (Dako) the sections were incubated with anti-mouse Alexa Fluor 488 (1:200; Life Technologies, Molecular Probes, Grand Island, NY, USA) or anti-rabbit rhodamine (1:200; Life Technologies, Molecular Probes) secondary antibodies for another 2 hrs. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies, Molecular Probes). Immunofluorescence studies were performed with a Zeiss Axio Imager Z1 microscope (Carl Zeiss MicroImaging GmbH) using 10×, 20×, 40×, and 63× objectives with the appropriate fluorescence filters. Digital images were acquired with a CV-M4+CL CCD camera (JAI, Yokohama, Japan) linked to a computer running the Isis software (Metasystems GmbH, Altlussheim, Germany).

For IHC, human samples were fixed in 10% buffered formalin and paraffin-embedded. 4 mm-tissue sections were deparaffinized and rehydrated. Novocastra Epitope Retrieval Solution (pH6 or pH9) was used to unmask antigens in a thermostatic bath at 98 °C for 30 min. Subsequently, the sections were brought to room temperature and washed in PBS. After neutralizing the endogenous peroxidases with 3% H₂O₂ and Fcblocking by 0.4% casein in PBS (Novocastra), the sections were incubated with primary antibodies for 90 min at room temperature. The immunostaining was revealed by a polymer detection method (Novolink Polymer Detection Systems Novocastra Leica Biosystems Newcastle Ltd Product No: RE7280-K) and 3,3’-diaminobenzidine (DAB) substrate-chromogen. Slides were analyzed under a Zeiss Axioscope A1 microscope and microphotographs were collected using a Zeiss Axiocam 503 Color digital camera with the Zen 2.0 Software (Zeiss). Slide digitalization was performed using an Aperio CS2 digital scanner (Leica Biosystems) with the ImageScope software (Aperio ImageScope version 12.3.2.8013, Leica Biosystems). Quantitative analyses of IHC stainings were performed by calculating the average percentage of positive signals in five nonoverlapping fields at medium-power magnification (X200) using Nuclear Hub or Positive Pixel Count v9 ImageScope software.

For IHC, human samples were fixed in 10% buffered formalin and paraffin-embedded. 4 mm-tissue sections were deparaffinized and rehydrated. Novocastra Epitope Retrieval Solution (pH6 or pH9) was used to unmask antigens in a thermostatic bath at 98 °C for 30 min. Subsequently, the sections were brought to room temperature and washed in PBS. After neutralizing the endogenous peroxidases with 3% H₂O₂ and Fcblocking by 0.4% casein in PBS (Novocastra), the sections were incubated with primary antibodies for 90 min at room temperature. The immunostaining was revealed by a polymer detection method (Novolink Polymer Detection Systems Novocastra Leica Biosystems Newcastle Ltd Product No: RE7280-K) and 3,3’-diaminobenzidine (DAB) substrate-chromogen. Slides were analyzed under a Zeiss Axioscope A1 microscope and microphotographs were collected using a Zeiss Axiocam 503 Color digital camera with the Zen 2.0 Software (Zeiss). Slide digitalization was performed using an Aperio CS2 digital scanner (Leica Biosystems) with the ImageScope software (Aperio ImageScope version 12.3.2.8013, Leica Biosystems). Quantitative analyses of IHC stainings were performed by calculating the average percentage of positive signals in five nonoverlapping fields at medium-power magnification (X200) using Nuclear Hub or Positive Pixel Count v9 ImageScope software.