Immediately after sacrificing the mice, tissue samples from the proximal, median and distal parts of the colon were collected and fixed overnight in 5% formalin. Paraffin inclusions were performed on the macroscopic injured area. Then, 4 µm sections were stained with hematoxylin-eosin-saffron (HES). A histological evaluation was performed by an experimented pathologist blinded to the nature of the mice being examined, using a five-degree severity score based on inflammatory cell infiltration, erosions and ulcerations, epithelial hyperplasia and mucin depletion, as previously described (29 (link)). Cytofix/Cytoperm buffer (eBioscience) was applied for the detection of nuclear staining. The primary antibodies used for immunostaining of mouse tissue and primary fibroblasts were: rabbit anti-mouse Ki-67 (clone SP6; Abcam), rabbit anti-human α-smooth muscle actin (α-SMA) (polyclonal; Abcam) and Alexa 488-conjugated rabbit anti-vimentin (Clone D21H3; Cell signaling). The secondary antibody was Alexa Fluor 555 goat anti-rabbit IgG (H+L) (Life Technologies). The nuclei were counterstained with DAPI (Sigma-Aldrich). Fluorescence analysis was performed with Olympus IX81 and Fluoview FV1000 software (Olympus). For quantitative analyses, at least four to five representative high-power fields (HPF, 40X) per section were evaluated in a blinded manner.
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