Plan apochromat 20x 0.8 m27

Microscopic evaluation, photography, and image processing were performed with a Zeiss Axio SlideScan Z.1 (Zeiss, Oberkochen, Germany) and the Zen Software 2.6 or Zen lite 3.5 (Zeiss) and the objective Plan- Apochromat 20x/0.8 M27, was used with the Orca Flash 4.0 V3 camera (Hamamatsu, Shizuoka, Japan). Exposure times of 2.07s, 601.3 ms, and 24 ms for Alexa Fluor 647, DyLight 550, and DAPI, respectively, were applied for all images equally to ensure comparability. In addition, image brightness editing was also applied equally for all sections. For automated image evaluation, the Zeiss Zen Image Analysis Software Add-On was used. Here, automatic segmentation for a region of interest (ROI) of 2000x1600 pixels was determined. The following settings were defined for the DAPI and 555 nm channel equally. Gaussian smoothing method and edge enhancement were chosen according to signal intensity and the threshold was adjusted and defined with the histogram. For optimal separation, the watershed method was applied. The size of the object was filtered with a minimum and maximum, after that a manual correction of software segmentation errors was performed blinded, such as separation of clustered signals, erasing of artifacts, and drawing of unrecognized events. Eosinophil counts were divided by nuclei counts of the ROI.