Immunohistochemistry (IHC) and immunofluorescence analyses were performed according to standard protocols. IHC was performed using a ready-to-use StreptAvidin Biotin-Complex (SABC) kit (Boster Biological Technology Co. Ltd., Wuhan, China). Sections were incubated with a rabbit anti-F4/80 antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA; 1:500 dilution), a rabbit anti-CK-19 antibody (Bioss; 1:200 dilution), a rabbit anti-amylase antibody (Bioss; 1:200 dilution), a rabbit anti-CD3 antibody (Bioss; 1:200 dilution), and a goat anti-COX-2 antibody (Santa Cruz; 1:200 dilution) for 48 h at 4°C. The corresponding secondary antibodies were subsequently incubated for 1 h at room temperature. Images were captured using a ZEISS Imager A1 microscope (Carl Zeiss AG, Oberkochen, Germany).
For double staining of immunofluorescence, a 1:200 dilution of COX-2 antibody and a 1:400 dilution of F4/80 antibody or CK-19 antibody was incubated in a humid chamber for 48 h at 4°C, and a 1:500 dilution of FITC-labeled anti-goat or CY3-labeled anti-rabbit secondary antibodies was incubated for 1.5 h. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Roche) for 5 min, dehydrated, and mounted on a coverslip with anti-fade mounting medium. Images were captured with a ZEISS Imager A2 fluorescence microscope (Carl Zeiss AG).
For double staining of immunofluorescence, a 1:200 dilution of COX-2 antibody and a 1:400 dilution of F4/80 antibody or CK-19 antibody was incubated in a humid chamber for 48 h at 4°C, and a 1:500 dilution of FITC-labeled anti-goat or CY3-labeled anti-rabbit secondary antibodies was incubated for 1.5 h. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Roche) for 5 min, dehydrated, and mounted on a coverslip with anti-fade mounting medium. Images were captured with a ZEISS Imager A2 fluorescence microscope (Carl Zeiss AG).