Immunofluorescence Staining of Tissue Sections

IF staining was performed as described previously (51 (link), 52 (link)). After blocking, the slides were incubated with the primary antibodies to Ln-γ2 (#ab210959, Abcam; dilution ratio, 1:500), pan-cytokeratin (#ab7753, Abcam; dilution ratio, 1:400), α-SMA (#19245, Cell Signaling Technology; dilution ratio, 1:200), CD3 (#ab1669, Abcam; dilution ratio, 1:150 dilution), CD4 (#ab183685, Abcam; dilution ratio, 1:200 dilution), CD8 (#ab217344, Abcam; dilution ratio, 1:200 dilution), EpCAM (#36746, Cell Signaling Technology; dilution ratio, 1:100), p-c-Jun (Ser⁷³) (#3270, Cell Signaling Technology; dilution ratio, 1:800), and p-c-Fos (Ser³²) (#5348, Cell Signaling Technology; dilution ratio, 1:200) at 4°C overnight in a moist chamber. After thorough washing, the slides were then incubated with Alexa Fluor 594– or 488–conjugated secondary antibodies (Invitrogen). Last, all slides were mounted with antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; #S36938, Thermo Fisher Scientific). Images were captured with an OLYMPUS FV2000 fluorescence microscope.