Ten days after air-pouch formation, substances were administered and after 8 h, mice were euthanized and their pouches were dissected, sampled and processed for paraffin embedding. The protocol for paraffin embedding was as follows (total 16 h): 70% ethanol (2 h), 80% ethanol (1 h), 95% ethanol (1 h), 100% ethanol (4.5 h), xylene (4.5 h), paraffin (58-60°C) (4 h), embedding tissues into paraffin blocks and trimming into the suitable 6 μm. Sections were then stained with hematoxylin (8 min) and eosin (1 min) (H&E) at 30°C, using the following protocol: The sections were deparaffinized, and treated with absolute alcohol (10 min), 95% alcohol (2 min), 70% alcohol (2 min) and stained with Harris hematoxylin solution (Thermo Fisher Scientific, Inc.) (8 min) and saturated lithium carbonate (1 min) at 30°C. After rinsing, the sections were counterstained with eosin-phloxine solution (Thermo Fisher Scientific, Inc.) (1 min) and dehydrated using 95% alcohol. Microphotographs were captured using a Nikon Eclipse 80i upright microscope with CFI60 lenses, using a Leica DFC 450 C digital camera. The magnification utilized for their capture was x200. In each section, pouch wall thickness was measured at 6 regions randomly at the upper, at the back and at the middle side of the pouch wall. The mean of six different regions in each section was determined. The thickness of the pouch and the area of the corresponding region were calculated using ImageJ software (v1.52a). The total cell number (based on nucleus count) was calculated as cells/mm2, using the 'analyze particles' feature of ImageJ software, in LPS-exposed mice bearing air-pouch with or without treatment, manually. The number of PMNs was calculated per mm2, in LPS-exposed mice bearing air pouch with or without treatment, manually.

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