Analysis of PMDZ dye retention in the colon

For the detection of hypoxia, mice were treated with 60 mg/kg of pimonidazole HCl (HP1-200Kit; Hypoxyprobe, USA) 30 min prior to sacrifice. Colon samples were fixed in 4% paraformaldehyde, and paraffin-embedded tissues were probed with mouse anti-pimonidazole monoclonal antibody (1:100; Hypoxyprobe) and then stained with Cy-3-conjugated goat anti-mouse antibody (1:50; SA00009-1, Proteintech), and then counterstained with DAPI using anti-fading mountant(S2110, Solarbio, China). Representative images were obtained using a Nikon fluorescent microscope (TE2000, Nikon, Tokyo, Japan).