Formalin-fixed, paraffin-embedded sections (4 µm) were mounted on Superfrost® Plus slides (Menzel-Gläser, Thermo Scientific) and dried for 30 min at 58°C. The sections were deparaffinized in xylene and rehydrated through decreasing concentrations of graded ethanol. The PT link system (100°C for 15 min in Tris/EDTA-buffer, pH 9.1) was used for epitope retrieval. Endogenous peroxidase activity was blocked by incubation in 3% H2O2 in methanol for 20 min at room temperature. Sections were then blocked in normal goat serum (1:50), diluted in phosphate-buffered saline (PBS) and incubated with the primary antibody S100A8 (1:3,200, Abcam) for 1.5 h at room temperature. Further steps were performed with EnVison + System-HRP AEC (Dako, K4009).
Washing between steps was performed with PBS for both procedures. Sections were counterstained in hematoxylin, and mounted using Aquatex medium (Merck). All runs included a negative control section in which the primary antibody was replaced with 1% PBS. The number of cells that were positive for the S100 calcium-binding protein A8 (S100A8) was quantified within the field of view of three representative areas in each animal/section (400× magnification, ø = 450 µm).
Washing between steps was performed with PBS for both procedures. Sections were counterstained in hematoxylin, and mounted using Aquatex medium (Merck). All runs included a negative control section in which the primary antibody was replaced with 1% PBS. The number of cells that were positive for the S100 calcium-binding protein A8 (S100A8) was quantified within the field of view of three representative areas in each animal/section (400× magnification, ø = 450 µm).