Whole-mount Immunofluorescent Staining of Ileal Tissue

Whole-mount immunofluorescent staining was performed using a modification of a previously reported method^{61 (link)}. The ileal tissue was fixed in 4% paraformaldehyde (Sigma) for 2 h at room temperature. Fixed tissue was permeabilized with 0.5% Triton X-100 (Sigma) overnight at room temperature, and then blocked with 10% goat serum (Sigma) and 0.5% Triton X-100 overnight at 4 °C. For SD and CDAHFD group, antibody reaction was performed with FITC labeled mouse anti-Crp1 antibody (50 μg/mL, clone 77-R63, produced in our laboratory) and Alexa Fluor 647-labeled anti-mouse/human CD324 (E-cadherin) antibody (1:100, clone DECMA-1, BioLegend). For CDAHFD + PBS and CDAHFD + R-Spo1 group, the primary antibody reaction was performed with rabbit anti-Olfactomedin 4 (Olfm4) antibody (1:80, clone D6Y5A, Cell Signaling) for 1 day at 4 °C, and then the secondary antibody reaction was performed with Alexa Fluor 555 conjugated F(ab′)2-goat anti-rabbit IgG (dilution 1:500, Thermo Fisher Scientific) and FITC labeled mouse anti-Crp1 antibody overnight at 4 °C. After washing, nuclei were stained with DAPI (Thermo Fisher Scientific). Samples were immersed in the optical-clearing solution (RapiClear 1.52, Sunjin Lab).
For quantification of Crp1 fluorescence intensity and counting numbers of Paneth cells and stem cells, Z-stack images were obtained using a confocal microscope (A1, Nikon) equipped with CFI Apo LWD 20X WI λS (Nikon). The number of Paneth cells was quantified by counting Crp1 immunostaining positive cells on 3 fields (150 × 150 μm²) per tissue. The number of stem cells was quantified counting Olfm4 positive cells on 3 fields (150 × 150 μm²) per tissue. Crp1 fluorescence intensity per Paneth cell was measured by creating a region of interest using image analysis software, NIS-Elements AR ver. 5.11 (Nikon), on 3 fields (150 × 150 μm²) per tissue, and the mean intensity per field was calculated. For quantification of the number and diameter of Paneth cell granules, Z-stack images were obtained using A1 with CFI Apo TIRF 60X Oil (Nikon). The number and diameter of Paneth cell granules were measured on 3 fields (33 × 33 μm², 2 Paneth cells/field) per tissue.