Strataquest analysis software

Tumor infiltrating neutrophils or intratumoral blood/lymphatic vessels in xenografts were stained immunohistochemically with anti-LY6G, anti-LYVE-1, or anti-CD31 antibodies and quantified by positive pixel count in relation to total tumor pixel count using Image Scope software, version 12.3.0.5056 (Leica, Wetzlar, Germany). Numbers of anti-LY6Gepositive tumor-infiltrating neutrophils in human tumors were counted with Strataquest Analysis software (TissueGnostics, Vienna, Austria). The distance of neutrophils to lymphatic vessels in xenograft sections was evaluated by immunofluorescent labeling with anti-LY6G, anti-LYVE-1, and DAPI using ZEN software (Zeiss) and classified into three categories ( 10, 11e20, and 21e200 mm).

Fluorescence imaging was performed with an Axio Imager 2 microscope (20×, Zeiss, Germany) and the TissueFAXS PLUS system (TissueGnostics, Austria). Images were processed and quantitatively analyzed with StrataQuest Analysis Software (v6, TissueGnostics). Optimized DAPI images were used to detect and segment nuclei (Figure 2). Nuclei areas were used to measure the mean staining intensities on FITC-and Cy5-shades in selected 1 mm 2 circular regions of interest (ROIs) that were placed around mesh fibers, each including about 2,000-5,000 nuclei (Figure 3). About 11 (median; range: 4-35) ROIs were analyzed in each sample for all stainings to represent the FBG. The area of the entire sample without the area of the ROIs was considered mesh-scar tissue. We recorded the total number of nuclei, as well as the percentages of FITC + (CD68 + ), Cy5 + (second marker + ), and the double-positive FITC + Cy5 + (CD68+ and second marker+) cells.
Additionally, controls without primary antibody and controls with isotype antibodies were performed, the later with all dilutions and incubation times (Figure 4). With a cut-off value of 100 for the mean intensity in the nuclear area on average less than 2% of false-positive cells were detected (Table 2).