Histopathological Scoring of Murine Colitis

Mouse colons were flushed with cold PBS to remove all contents, and the entire colon was rolled into Swiss rolls. The tissues were fixed in 2% PFA for 12 h at 4 °C and then washed in 1x PBS and transferred to 70% ethanol until embedding. Swiss rolls were embedded in the same manner as described above for segments of mouse colon. Paraffin-embedded, PFA fixed tissues were sectioned at 4.5 μm. Tissue sections were de-paraffinized and hematoxylin and eosin stained (Mayer’s Hematoxylin, Thermo Scientific; Eosin 1%, Morphisto, Germany). Histopathological analyses were performed using a semi-quantitative scoring system^{78 (link)} that evaluated the severity of crypt damage and cellular infiltration, epithelial erosion and tissue thickening using a severity score from 0 to 3 (0 = intact, 1 = mild, 2 = moderate, 3 = severe), and those scores were multiplied by a score for percent involvement (0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 50–100%). A trained and blinded scientist performed the scoring. Representative images were acquired using an Olympus CKX53 microscope and Olympus SC50 camera.