Fluorescence imaging was conducted using an Axio Imager 2 microscope (20×, ZEISS, Jena, Germany) equipped with a Colibri 7 light source (ZEISS, Germany) and the TissueFAXS PLUS system (TissueGnostics, Vienna, Austria). The light source comprises six LED modules and seven fluorescence channels, each emitting monochromatic light of a different wavelength. LED-optimized filters and direct coupling enhance sensitivity and ensure optimal excitation and emission spectra.
The images were analyzed using StrataQuest Analysis Software (v7, TissueGnostics, Vienna, Austria), and the minimum and maximum ranges for each filter were set by automatically adjusting the saturation. The mean minimum and maximum intensities of the slides were used for each marker in each panel [22 (link)].
Nuclei were detected and segmented using DAPI images. The mean staining intensities for the five different markers were measured using nuclei areas in six equally distributed circular ROIs with an area of 0.785 mm2 (a circular ROI that fits into a 1 × 1 cm square) per slide. The total number of cells with a mean intensity greater than 100, which were considered as ‘positive’, was recorded.
The images were analyzed using StrataQuest Analysis Software (v7, TissueGnostics, Vienna, Austria), and the minimum and maximum ranges for each filter were set by automatically adjusting the saturation. The mean minimum and maximum intensities of the slides were used for each marker in each panel [22 (link)].
Nuclei were detected and segmented using DAPI images. The mean staining intensities for the five different markers were measured using nuclei areas in six equally distributed circular ROIs with an area of 0.785 mm2 (a circular ROI that fits into a 1 × 1 cm square) per slide. The total number of cells with a mean intensity greater than 100, which were considered as ‘positive’, was recorded.
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