Immunostaining of Stomach Tissue Sections

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).