Aperio cs2 digital scanner

Stained slides were acquired using the Aperio CS2 digital scanner and ScanScope software (Leica Biosystems, Wetzlar, Germany). Images were viewed and organized using ImageScope software (Leica biosystems, Wetzlar, Germany). Region of interest consisted of the tumor area and normal colon; necrotic areas were excluded from the selection. IHC Nuclear Image Analysis algorithm (Leica biosystems, Wetzlar, Germany) was setup for the analysis to identify categories of strong and weak positive cells based on the signal intensity (Supplementary Fig. 18). Data are expressed as the absolute number of NOVA2-positive cells per mm² and as fractions of strong and weak positive cells.

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.