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Animal experiment: Inflammatory cell response evaluation1656isopropanol) and infiltration with xylene and paraffin in a Leica Peloris 3 infiltration automaton (Deer Park, IL, USA). After processing all tissues were embedded in paraffin and stored at 4°C. All fixed and paraffinembedded (FFPE) specimen were cut on a Leica RM 2255 rotation microtome (Deer Park, IL, USA), including a cooling unit and a water basin. Five µm thin sections were put onto special adhesive microscopic SuperFrost Plus slides (VWR Collection, Darmstadt, Germany) dried overnight and then stored at 4°C until histologic processing. A hematoxylin-eosin coloring was then applied to these tissues.(Table 1) While hematoxylin is a nuclear stain that results in a purple to blue color after processing, eosin is a cytoplasmic stain. It results in a bright pinkishred color in red blood cells; muscle fibers; collagen fibers and was used to evaluate the tissue, including the inflammatory cells present. Analysis of the slides was performed using a light microscope (BX40 (Olympus Belgium N.V., Antwerp, Belgium)) at a magnification of 4x, 10x, 20x and 100x.Table 1: Hematoxylin & Eosin staining protocol Step Reagent/solution Time 1 Xylene 0:05:00 Deparaffinization2 Xylene 0:05:00 Deparaffinization3 Ethanol 96% 0:05:00 Rehydration4 Ethanol 80% 0:05:00 Rehydration5 Ethanol 70% 0:05:00 Rehydration6 Aqua dest 0:01:30 Rehydration7 Hematoxylin 0:05:00 Staining cell nuclei8 Aqua dest 0:00:30 Wash/removal of excess staining solution9 Running water 0:05:00 Wash/Blueing of hematoxylin (with fixation of the hematein molecules)10 Eosin 1%, aqueous, pH 6 0:05:00 Staining of cytoplasm and other components11 Running water 0:04:00 Wash12 Ethanol 96% 0:01:30 Dehydration13 Ethanol 96% 0:02:00 Dehydration14 Isopropanolol 0:05:00 Dehydration15 Xylene 0:05:00 De-alcoholization16 Xylene 0:05:00 De-alcoholizationNikolas de Meurechy NW.indd 165 05-06-2024 10:14