Dm5500

Sections from formalin-fixed, paraffin-embedded proximal intestinal segments (S1) were deparaffinized, rehydrated, and stained with hematoxylin (Sigma-Aldrich, Cat #MHS16-500 ML) and eosin (Sigma-Aldrich, Cat #HT110332-1L) then imaged on a brightfield microscope (Leica DM5500, Buffalo Grove, IL). Histological markers of intestinal adaptation were measured from representative images of a single intestinal section for each fish, including villus height (VH) and villus epithelial perimeter (VEP), as validated in our prior work [18 (link)]. Only complete villus folds were included in analysis. All measurements were performed with ImageJ software (NIH.gov) and are reported in μm±SEM.
Quantification of epithelial cell proliferation and epithelial cell death with immunofluorescent staining for BrdU and cleaved caspase-3 (CC3).
Proximal intestinal sections were stained for BrdU and CC3 as described [14 (link),18 (link)]. Images were taken with a Leica immunofluorescent microscope (Leica DM1600B, Buffalo Grove, IL). Staining was quantified by counting the number of positively staining epithelial cells per villus fold and reported as BrdU + cells/villus fold ±SEM or CC3+ cells/villus fold±SEM.

Adult human RPESC-RPE on 24-well size Transwell inserts (Corning) were fixed with 4% paraformaldehyde for 10 min, rinsed 3× with phosphate-buffered saline, permeabilized with 0.02% Tween 20, and blocked with normal goat or donkey serum (5%) in BSA (1%) for 1 h. Primary antibodies against SNAI1 (goat, diluted 1:100, R&D Systems, cat. no. AF3639), Z-O1 (mouse, diluted 1:100, Invitrogen, cat. no. 339100), and CLDN19 (rabbit, diluted 1:100, Abcam, cat. no. ab74374) were added, incubated overnight at 4°C, then washed and incubated for 45 min at room temperature with the corresponding Alexa Fluor-conjugated secondary antibodies (1:1,000) (Life Technologies Alexa Fluor 488 donkey anti-goat IgG [(H + L], cat. no. A11055; Alexa Fluor 488 goat anti-mouse IgG [H + L], cat. no. A11017; Life Technologies Alexa Fluor 488 donkey anti-rabbit IgG [H + L], cat. no. A11070). DAPI (Roche, cat. no. 10-236-276-001) was added at room temperature for 45 min as a nuclear counterstain. Cells on Transwell inserts were mounted on glass slides with Prolong Gold (Life Technologies, cat. no. P36930) and imaged by fluorescent microscopy (Leica DM5500). Western blot methods are provided in Supplemental Information.

Transfected or untransfected CSE-treated 16HBE cells (1 × 10³/well) were plated into each well of 96-well plates and incubated for an additional 2 h in the respective medium containing 50 μM EDU (RiboBio Guangzhou, China). Then, the cells were fixed with 4% formaldehyde and stained with Dye solution (RiboBio). After washing with 3% bovine serum albumin (BSA), cell nuclei were stained with DPAI. Images were visualized with fluorescence microscopy at 100× magnification (Leica DM5500, Germany). Triplicate individual experiments were performed in this study.