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For protein extraction, each tissue was placed into a tube containing a lysis buffer consisting of 20 mM Tris HCl with pH 7.6, 0.5 M Sucrose, 0.15 M KCl, 20 mM DTT supplemented with 100 L/mL of a protease inhibitor cocktail (Sigma-Aldrich, MO, USA) in a 1:3 (v/v) ratio

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However, it has become clear that the most widely used enzyme-linked immunosorbent assays (ELISA) do not detect zonulin but complement C3 and possibly properdin, a related molecule from the same family with unclear functional effects (73, 74)

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