Immunohistochemical and Immunofluorescence Analysis

4 μm thick paraffin embedded tissue sections were deparaffinized, and antigen-retrieval was performed using Target retrieval solution (Dako, Carpinteria, CA) in Decloaking chamber. Sections were then blocked with 1%BSA and incubated with anti-pSTAT3-Y705 (Abcam), anti-F4/80 (Abcam) or anti-Ki-67 (Abcam) overnight at 4°C. The sections were then incubated with anti-rabbit-horseradish peroxidase–labeled polymer (Dako) for 1 hour at room temperature, followed by DAB substrate (Dako), counterstaining with hematoxylin, and mounting with Permount mounting medium (for immunohistochemistry). For double-immunofluorescence, after antigen-retrieval and blocking with 1%BSA, staining was performed with anti-Foxp3 (ebiosciences) and anti-RORγT (Abcam), or anti-CD8 (Abcam) and anti-Lamp1 (Abcam). Antibodies were applied on tissue sections overnight at 4°C, followed by anti-rat Alexaflor-594 and anti-rabbit Alexaflor-488 for 1 hour in the dark at room temperature followed by DAPI (Invitrogen), and mounted as described [21 (link)].