Eosinophil Quantification in Tissue Sections

Sections of the distal colon and jejunum were fixed overnight in paraformaldehyde 3.7 % (Sigma-Aldrich, Diegem, Belgium) and embedded in paraffin. Sections of 5 µm were cut with a microtome. Specific quantification of eosinophils was performed using Chromotrope 2R staining [16 (link),19 (link)]. Briefly, following deparaffinization with xylene and rehydration with serial ethanol dilutions, slides were immersed for 1.5 h in a solution containing Phenol (106.2 mM; Sigma-Aldrich, Diegem, Belgium) and Chromotrope 2R (21.3 mM; Sigma-Aldrich, Diegem, Belgium). A counterstaining was performed by the application of hematoxylin during 15 s on the sections. The number of positive cells was counted in 5 nonoverlapping high-power fields per slide, one slide per animal, and expressed as the number of chromotrope-2R-positive cells per area of lamina propria.