Dab substrate kit

Xenograft tumors were harvested from the mice, fixed in 4% paraformaldehyde, embedded into paraffin, and then cut into 4-μm-thick sections. Sections were stained with hematoxylin. The slides were blocked with goat serum at 37°C for 20 min and incubated with the primary antibody (1:100, Cell Signaling Technology, Cambridge, UK) at 4°C overnight. Then, each sample was incubated with the secondary antibody (Cell Signaling Technology, Cambridge, UK) for 30 min at room temperature, followed by staining with a DAB substrate kit (Agilent Technologies, Santa Clara, CA). After dehydrating and drying, the stained samples were observed under a microscope. The signals were analyzed with the ImageJ software.
The expression levels of cytokines including IL-10, IFN-γ, and TNF-α in mice serum were detected using ELISA kits (Sino Biological, Beijing, China). All the procedures of ELISA assays followed the manufacturer’s protocol.

The cholesteatoma and RA skin tissues were fixed in 4% paraformaldehyde. Tissues were snap frozen and embedded in OCT compound (Sakura Finetek, Torrance, CA, USA). Tissues were sectioned at a thickness of 10 μm and mounted on slides (Matsunami, Osaka, Japan). Tissue sections were digested with proteinase K (Agilent, Santa Clara, CA, USA) for 15 minutes at room temperature, followed by being washed with phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 15 minutes, followed by being washed with PBS. Subsequently, tissue sections were incubated with non-specific staining blocking reagent (Nacalai tesque, Kyoto, Japan) for 20 minutes at room temperature, and incubated with 200x diluted primary rabbit anti-CYLD polyclonal antibody (Sigma, St. Louis, MO, USA) overnight at 4°C. Then, tissue sections were incubated with secondary antibodies for 60 minutes at room temperature, followed by being washed with PBS. For visualization, DAB Substrate Kit (Agilent) was used according to the manufacturer’s instructions, followed by counterstaining with Mayer’s hematoxylin (Wako, Osaka, Japan).

Tissue sections were processed as above and were stained with antibodies specific for CK5 (rabbit, 1:200, ab75869, Abcam, Cambridge, UK) and CK17 (rabbit, 1:100, ab109725, Abcam) for 2 h at RT. Sections were blocked using HRP Blocking Reagent (Abcam) EnVision+/HRP Visualization (Agilent, Santa Clara, CA, USA) and DAB substrate kit (Agilent) were used to visualize staining. Sections were counterstained with hematoxylin and mounted with Permount Mounting Medium (Fisher Scientific, Waltham, MA, USA). Representative photographs were taken under a light microscope at ×20 magnification.