Gut Permeability and Tight Junction Assessment

Gut permeability was determined by fluorescein isothiocyanate dextran (FITC-dextran) assay, endotoxemia, and immunofluorescent detection of a tight junction protein (zonaoccludens-1; ZO-1) following previous publications^{33 (link)–35 (link)}. As such, FITC-dextran, a nonabsorbable molecule with 4.4 kDa molecular mass (Sigma-Aldrich, St. Louis, MO, USA) at 12.5 mg per mice was orally administered at 3 h before the detection of FITC-dextran in serum by Fluorospectrometer (NanoDrop 3300; Thermo Fisher Scientific, Wilmington, DE, USA). Serum endotoxin (LPS) was measured by HEK-Blue LPS Detection (InvivoGen, San Diego, CA, USA) and the data were recorded as 0 when LPS values were less than 0.01 EU/ml because of the limited lower range of the standard curve. Also, the cecum was used as a representative of the intestine to determine gut tight junction. Accordingly, cecum in Cryogel (Leica Biosystems, Richmond, IL, USA) were cut into 5 μm-thick frozen sections, fixed in acetone, blocked by blocking buffer, stained with a fluorescent antibody against ZO-1 and a green secondary antibody (Alexa Fluor 488) (Life Technologies, Carlsbad, CA, USA) before visualization and scoring by ZEISS LSM 800 (Carl Zeiss, Germany).