Figure legends
Figure 1: Displayed representative images show 3D-reconstructions of triple-immunostaining in 100 µm biopsy sections for EG2+ surfaces as a marker for eosinophils (red surfaces), NE+ surfaces as marker for neutrophils (yellow surfaces) and cytokeratin (green surfaces) at baseline (A) and 24 hrs after segmental allergen provocation (B) of one subject; Settings for image acquiring: x25-water objective; argon laser (488 nm), helium/neon1 laser (543 nm) and helium/neon2 laser (633 nm) ; 90 optical slices with an interval of 1.1 µm; grid spacing: 50 µm; C-D: Depicted are the graphs of the quantification of volume of EG+ surfaces [µm/100000 µm3] (C) and volume of NE+ surfaces [µm/100000 µm3] (D) before (Baseline) and 24 hrs after segmental allergen provocation (Allergen). Each symbol represents one subject (for symbol legend refer to tab. 1). Per subject one 100 µm biopsy section comprised of 4 to 7 regions was analysed. The groups were compared using Wilcoxon matched-pairs signed rank test; **: p < 0.01, number of subjects: C: n = 10; D: n = 10; AL = Airway lumen; EG2 = antibody used to identify ECP; NE= neutrophil elastase; SAP = segmental allergen provocation.
Figure 2: Images of a granule filled cell positive for EG2 (red) displayed in the fluorescent channel (A), transmitted light channel (B) and as a merge of both channels (C); white circle: outline of cell, white disrupted circle: cell nuclei; D: distribution of EG2+ staining within a tissue volume of a 20 µm thickness; E: 3D-reconstruction of the stack image shown in D; white stars: EG2+ staining/surfaces resembling cellular structure; white small circles: EG2+ staining in free granule structures; thin white arrows: EG2+ staining distributed within the tissue unbound; F – H: Images show 3D reconstruction of 100 µm biopsy sections of three different subjects 24 hrs after segmental allergen provocation. The distribution of EG2+-staining (red surfaces) beneath the epithelial layer (green surfaces) is very diverse between the subjects. Settings for image acquiring A - E: x40-water objective; helium/neon1 laser (543 nm); grid: 10 µm; scale bar: 10 µm; F – H: x25-water objective; argon laser (488 nm) and helium/neon1 laser (543 nm); 90 optical slices with an interval of 1.1 µm; grid spacing: 50 µm; AL = airway lumen; EG2 = antibody used to identify ECP; SAP = segmental allergen provocation.