Intestinal Morphometric Analysis Protocol

Five intestinal samples were randomly selected from each group and processed according to the method described by Thompson et al. [29 (link)]. In brief, the intestinal samples were collected from the distal (about 1 cm from the colon). Intestinal samples were dehydrated with increasing concentrations of ethanol, cleared with xylene (Surgipath Medical Industries, Richmond, IL), and embedded in paraffin wax (Thermo Fisher Scientific, Kalamazoo, MC). Cross-Sect. (5 μm) were stained with hematoxylin and eosin (GeneCopoeia, Rockville, MD). The stained sections were dehydrated with ethanol, cleared with xylene, and mounted with DPX mountant based on the method of Jiang et al. [30 (link)]. ImageJ software (National Institutes of Health, USA) was used to determine the morphometric measurements of villus height and crypt depth of the cecal using an Olympus BX40 F-3 microscope (Olympus Cooperation, Tokyo, Japan) attached to a digital video camera (Q-imaging, 01-MBF-200R-CLR-12, SN: Q32316, Canada) [31 (link)].