The tissue slices (4 μm) were deparaffinized with xylene and rehydrated in a graded alcohol series and distilled water. After blocking the endogenous peroxidase with hydrogen peroxide, citrate buffer (ZhongShan Golden Bridge Biotechnology Co., Ltd) was used to perform antigen retrieval in water bath at 95°C for 35 minutes. After naturally cooling down, the slices were incubated with primary monoclonal antibodies to CD54 (1:75, Abcam), Lgr5 (1:60, Abcam), Oct4 (1:200, Abcam), EpCAM (1:800, Abcam), Sox2 (1:200, ProMab) at 4°C and with primary monoclonal antibody to CD133 (1:10, Miltenyi Biotec) at room temperature overnight. For immunofluorescence, the frozen slices (8 μm) of GCSCs spheres were incubated with these primary monoclonal antibodies under the same conditions, except EpCAM (1:200, Abcam). Subsequently, these slices were incubated with peroxidase-conjugated polymer (EnVision™ Detection Kit, Gene Tech (Shanghai) Company Limited) for 30 minutes or fluorescent secondary antibody (1:800, Alexa Fluor 555) for 90 minutes at room temperature. Finally, the slices were stained with diaminobenzidine chromogen solution (1:50, EnVision™ Detection Kit, Gene Tech (Shanghai) Company Limited) and counterstained with hematoxylin (ZhongShan Golden Bridge Biotechnology Co., Ltd) or DAPI (1:4000). Primary antibody incubation was omitted in negative controls. The specificity of all antibodies was demonstrated by providers. The figures were captured through Axio Imager A2 (Zeiss) and Scope A1 (Zeiss). As reported previously, GCSCs were cultured in serum free DMEM/F12 medium supplemented with 20 ng/ml EGF and 10 ng/ml b-FGF .