Alexa fluor 637

Mice under deep anesthesia were transcardially perfused with ice-cold PBS, followed by immersion in 4% paraformaldehyde. Coronal sections were prepared, blocked and then cut. Frozen sections were incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-PDGFR-β (1:50, Cell Signaling Technology), mouse monoclonal anti-NG2 (a marker of cerebrovascular pericyte 1:100, Santa Cruz), rabbit polyclonal anti-TGF-β (1:100, Abcam), rabbit polyclonal anti-Ki-67 (1:100, Cell Signaling Technology), rabbit polyclonal anti-MCP-1 (1:200, Abcam) and rabbit polyclonal anti-TNF-α (1:50, Abcam). Then, the sections were incubated with appropriate host secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 637 (1:200, Invitrogen, USA). All sections were stained with 6-diamidino-2-phenylindole (DAPI, CA, USA) and photographed with a confocal microscope (TCS SP5, Leica).

The small intestines from mice were divided into approximately 1-cm portions, and each portion was opened with a longitudinal cut. After several washes with PBS, intestines were fixed with 4% PFA for 2 h and then perfused with 10% sucrose. Intestinal sections were embedded in optimal cutting temperature (OCT) compound and frozen to −80°C. A cryostat was used to cut 5- to 7-μm-thick transverse intestine slices, which were mounted onto microscope slides. The slices were washed with PBS, incubated with 2 N HCl for 30 min, and washed three times with 0.1 M Na₂B₄O₇ (pH 9). For BrdU and ZO-1 labeling, the slices were permeabilized with PBS–0.1% Triton X-100–10% horse serum for 30 min and then incubated with anti-BrdU (Abcam; Ab 1893) and anti-ZO-1 (Zymed Laboratories 33-9100), followed by anti-sheep Alexa Fluor 637 and anti-mouse Alexa Fluor 488 secondary antibodies (Invitrogen), respectively, or phalloidin (Invitrogen). Immunohistochemistry images were acquired in a blind manner for BrdU, as images were selected by DAPI (4′,6-diamidino-2-phenylindole) and ZO-1 staining. Samples were all imaged using a confocal laser scanning microscope (LSM510 or LSM710; Zeiss).