Colon sample (2–3 cm up anus) were first fixed in 10% Formalin (Bio-Optica Milano S.p.A. Milan, IT), embedded in Paraffin (Bio-Optica Milano S.p.A. Milan, IT), cut into 5-μm-thick sections and then stained with Hematoxylin/Eosin (H&E) (Bio-Optica Milano S.p.A. Milan, IT) for histopathological analysis. The histological score of the colon was assessed as previously described by U. Erben et al. [29]. This score evaluated the level of tissue inflammation in relation to the extension of the inflammatory cell infiltrate (mild severity if the cellular infiltrate is only present in the mucosa, moderate if they are involved mucosa and submucosa, marked if the infiltration is transmural), and epithelial changes of the intestinal mucosal architecture like erosions and ulcerations.

Immunohistochemistry was performed on paraffin embedded colon. In brief, Ag retrieval was achieved by incubation of the slides for 90 min in the hot (95 °C) sodium citrate buffer (pH 6.0) and 30 min of cooling at room temperature. Immunostaining technique was performed using the commercial kit Elabscience®2-step plus Poly-HRP Anti Rabbit/Mouse IgG Detection System (with DAB Solution) (Houston, Texas 77079, USA.) ACE2 Recombinant Rabbit Monoclonal Antibody (SN0754) Invitrogen, Thermofisher scientific (Waltham, Massachusetts, USA) was incubated overnight at 4˚C. Subsequently, sections were incubated with Polyperoxidase-anti-Mouse/Rabbit IgG and then with DAB Working Solution, both supplied by the kit. Slides were counterstained with hematoxylin, dehydrated through ethanol and xylene, and coverslipped using a xylene-based mounting medium.

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