Paneth Cells

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.

Crypts were isolated from the small intestine of mice fed with SD or CDAHFD for 3 wk as previously described^{17 (link)}. Briefly, the ileum segments were shaken in cold HBSS containing 30 mM EDTA. After vigorous shaking for ~ 300 times in HBSS, the isolated crypts were resuspended in HBSS containing 300 U/mL collagenase (Sigma), 10 µM Y-27632 (Sigma), and 1 mM N-acetylcysteine (Sigma), and shaken at 180 rpm for 5 min at 37 °C on a horizontal shaker (TAITEC). Then, 50 μg/μL Dnase I (Roche) was added, and the sample was mixed by pipetting. Cells were pelleted at 500 g for 5 min at 4 °C and resuspended in washing buffer (DMEM/F12 containing 10 μM Y-27632 and 1 mM N-acetylcysteine), then passed through 40-μm cell strainer (BD Falcon). Paneth cells were stained with Zinpyr-1 (Santa Cruz) and Allophycocyanin (APC) anti-CD24 (clone M1/69,)Abcam) in washing buffer for 10 min at 37 °C. After Paneth cell labeling, the cells were sorted by flow cytometry using a cell sorter (JSAN, Bay Bioscience). Single cells were gated by forward scatter and side scatter. Cells were sorted directly into lysis buffer for RNA isolation (PureLink RNA Mini Kit, Invitrogen). To make a pooled sample, at least 10,000 Paneth cells were sorted from 2 separate mice, and sorting experiment was repeated three times. 2 pools (each pool contains Paneth cells from 6 separate mice) were prepared for each group. Total RNA was isolated using Invitrogen® PureLink RNA Purification System according to manufacturer’s instructions, and cDNA was synthesized using a SMART-Seq. Sequencing libraries were built with the TruSeq RNA Library Prep Kit (Illumina) and then submitted to Illumina NovaSeq 6000 for 100-bp PE reads sequencing. Fragments per kilobase of transcript per million mapped reads (FPKM) values were used, genes with FPKM values below 1 were not included in the analysis, and fold change ≥ 1.5 or ≤ 0.67 was considered differentially expressed.