Hoechst dye 33258

Rats were deeply anesthetized using isoflurane and perfused transcardially with 4% formaldehyde buffered with 0.01 M phosphate-buffered saline (PBS; pH 7.4). Skin samples were removed, postfixed in 4% formaldehyde solution in 0.01 M PBS at 4 °C overnight, and immersed in 30% sucrose solution. Skin sections (40-μm thickness) were prepared using a cryostat and collected as free-floating sections. For double-immunostaining, the skin sections were incubated with several primary antibodies at 4 °C for 12–18 h; these antibodies were as follows: monoclonal mouse anti-NG2 IgG (1: 200, Millipore), polyclonal rabbit anti-NG2 IgG (1: 200, Millipore), polyclonal rabbit anti-Ki67 IgG (1: 1,000, NovoCastra), polyclonal rabbit anti-Lgr5 (GPR49) IgG (1: 100, Abcam), polyclonal rabbit anti-CD34 IgG (1: 150, Abcam), polyclonal rabbit anti-cytokeratin 15 (CK15) IgG (1: 100, Abcam), polyclonal rabbit anti-Gli1 IgG (1: 200, Abcam), polyclonal goat anti-luciferase IgG (1: 50, Promega). After washing for 30 min with PBS containing 0.3% Triton-X100 (PBST), skin sections were incubated with the appropriate secondary antibodies conjugated with either Cy2 or Cy3 (1: 200, Jackson ImmunoResearch) at 4 °C for 3–4 h, and washed with PBST for 30 min. The stained sections were mounted with Hoechst dye 33258 (Nacalai Tesque Inc.) and examined using a confocal laser microscope (Digital Eclipse C1; Nikon).

Immunostaining studies were performed as described previously [22 (link)]. Stomach tissues were removed and post-fixed in a 4% formaldehyde solution in 0.01 M PBS at 4°C for 2 h, then soaked in 30% sucrose solution. Stomach sections (40 μm thick) were prepared using a cryostat microtome and incubated with monoclonal mouse anti-NG2 IgG (1:100, Abcam, Cambridge, MA, USA), polyclonal rabbit anti-NG2 IgG (1:100, Abcam), monoclonal rat anti-CD34 IgG (1:100, Santa Cruz, CA, USA), polyclonal rabbit anti-PDGFRα IgG (1:200, Abcam), polyclonal rabbit anti-PDGFRβ IgG (1:100, Santa Cruz), monoclonal mouse anti-CD31 IgG (1:100, BD Biosciences, New Jersey, USA), monoclonal mouse anti-αSMA IgG (1:100, Abcam), or polyclonal goat anti-luciferase (Luc) IgG (1:50, Promega, Madison, USA) antibodies at 4°C for 15–20 h. After washing for 30 min with 0.3% Triton-X100 in PBS (PBST), the sections were incubated with the appropriate Cy2, Cy3, or Cy5-conjugated secondary antibodies (1:200, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at 4°C for 2–4 h and washed with PBST for 30 min. The sections were mounted with Hoechst dye 33258 (Nacalai Tesque, Kyoto, Japan.) and observed using a confocal laser microscope (Digital Eclipse C1; Nikon, Tokyo, Japan).