Quantifying Paneth Cells and Proliferation

The terminal part of the small intestinal tissue was harvested, fixed in formalin (#133-10311; FUJIFILM Wako), and embedded in paraffin. Using the sections of hematoxylin–eosin staining, three intact crypts were randomly selected from each sample, and Paneth cells were manually counted under a microscope (400× magnification) by a researcher who was blinded to the mouse genotypes. For immunohistochemistry, the following primary antibodies were used: Ki67 (1:100; #12202; Cell Signaling) and LAT1 (provided by Osaka University [19 (link)]). The secondary antibody #K4003(Dako) was used for Ki67 staining. For LAT1 staining, the ABCHRP Kit Peroxidase (#pk6101; Vector Laboratories) was used. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for the analysis of Ki67-positive cells by two researchers who were blinded to the mouse genotypes.