Carnoy s solution

Colonic tissues were fixed in Carnoy’s solution (Wako) to preserve the mucus layer, embedded in paraffin, and cut to 5 µm. The sections were hybridized to a mixture of three Bacteroidetes-specific probes labeled with Cy3. The sequences of the probes are (1) 5′-CATTTGCCTTGCGGCTA-3′ (Momose et al., 2011 (link)), (2) 5′-CCAATGTGGGGGACCTT-3′ (Manz et al., 1996 (link)), and (3) 5′-AGCTGCCTTCGCAATCGG-3′ (Weller et al., 2000 (link)). Since phylum Bacteroidetes was efficiently depleted by metronidazole and ciprofloxacin (Bloom et al., 2011 (link); Fig. S4 C), this strategy specifically visualized B. theta in SPF-Abx mice. Images were acquired on a Zeiss LSM 710 confocal laser-scanning microscope. The bacterial length was measured with Adobe Photoshop CS 5.1 on acquired images.

The distal half of the colon tissue was harvested, fixed with 10% (w/v) Formaldehyde Neutral Buffer Solution (Nacalai, Kyoto, Japan) overnight, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E) and with Periodic acid–Schiff (PAS). Crypt-cell proliferation and tight-junction were determined by immunohistochemistry with anti-Ki67 antibody (#652402, Biolegend, San Diego, CA) and anti-ZO-1 antibody (#GTX108592, Genetex, Irvine, CA), respectively. Apoptosis was detected by TUNEL assays, using the In Situ Cell-Death Detection Kit, peroxidase (POD) (Roche, Basel, Switzerland). For Alcian blue/PAS staining, the distal colon was harvested, submerged in Carnoy’s solution (Wako, Japan) at 4 °C for 2 h, and placed into 100% ethanol. The fixed colon tissues were embedded in paraffin, cut into 5 μm sections, and stained with Alcian blue/PAS. Microscopic images of the organoid cultures were obtained with a BZ-X700 (Keyence, Osaka, Japan).