Pe vectra

Tissue multicolor immunofluorescent staining was performed using the OpalTM 7‐Color Manual IHC Kit (NEL811001KT, Perkinelmer). Tumor tissues were fixed and embedded in paraffin and sectioned with a microtome. The sections were routinely dewaxed and hydrated. Tris‐EDTA Buffer solution was applied for antigen retrieval, 3% H₂O₂ was used to quench endogenous peroxidases, and samples were blocked in normal goat serum. The slides were then incubated with Brilliant Violet 421 anti‐mouse CD206 (MMR) Antibody (Biolegend, cat# 141717) and Alexa Fluor 488 anti‐mouse F4/80 Antibody (Biolegend, cat# 123119) overnight. Next, they were incubated with DAPI for 1 h at room temperature. Finally, tissue immunofluorescence was analyzed using the PE Vectra (Perkinelmer).

Tissue multicolor immunofluorescent staining was performed by Opal^TM 7-Color Manual IHC Kit (NEL811001KT, Perkinelmer). Tumor tissues were fixed and embedded in paraffin and sliced with a slicer. The slices are routinely dewaxed and hydrated. Tris-EDTA Buffer solution for antigen repairing, 3% H₂O₂ for inactivation of endogenous peroxidase, and normal goat serum for blocking. The slides were then incubated with CD8 (A02236-1), CD86 (ab213044), CD206 (ab64693), F4/80 (ab6640), and IFN-γ (ab9657) overnight. Next, they were incubated with HRP labelled goat anti-rabbit / mouse secondary antibody in combination with DAPI for 1 h at room temperature. Finally, tissue immunofluorescence was analyzed using PE Vectra (Perkinelmer). CD8 antibody was purchased from BOSTER Biological Technology Co., Ltd (Wuhan, China). The antibodies against CD86, CD206, F4/80, and IFN-γ were purchased from Abcam.