Immunohistochemistry (IHC) and immunohistofluorescence (IHF)

IHC and IHF were performed as previously reported.¹⁸ Briefly, tumor section (4 μm) were dewaxed in xylene, hydrated in decreasing concentrations for 30 min, washed with phosphate-buffered saline, and probed with monoclonal antibodies or isotype controls at 4°C overnight. The primary antibodies used were the same as the ones mentioned above. After being washed, the sections were incubated with biotinylated goat anti-rabbit or anti-mouse IgG at room temperature for 2 h. Immunostaining was visualized with streptavidin/peroxidase complex and diaminobenzidine, and sections were counterstained with hematoxylin. Slides were visualized under a bright-field microscope at ×40 and ×400 magnification. Immunofluorescence staining images were taken by ZEISS microscope (LSM880, Germany). Positive cells were quantified using ImagePro Plus software (Media Cybernetics) and expressed as mean ± SEM in high-powered fields detected by confocal microscopy.