Streptavidin tagged horseradish peroxidase

All IHC experiments were performed with the formalin‐fixed, paraffin‐embedded sample. Paraffin blocks were cut into 5 μm thick sections, mounted onto poly‐L‐lysine‐coated slides, and dried. After the dried slides were de‐paraffinized, antigen retrieval was performed for 20 minutes by using an automated antigen retrieval machine in the presence of ethylenediaminetetraacetic acid (pH 9.0). Non‐specific binding to the sections was blocked by incubation in 10% normal goat serum (Gibco Life Technologies) for 1 hour prior to incubation with the VLDLR (Cat.no. sc‐18824) and iNOS (Cat.no. sc‐650) primary antibodies overnight at 4℃. Secondary mouse antibodies were used to detect primary antibodies, followed by incubation with streptavidin‐tagged horseradish peroxidase (Ventana Medical Systems). Diaminobenzidine (Sigma‐Aldrich) was used to induce signalling. The IHC slides were visualized with an optical microscope (Leica) and analysed with Leica software.

All IHC was performed on formalin-fixed, paraffin-embedded samples. Paraffin blocks were sectioned to 5-μm thickness. Afterwards, poly-L-lysine-coated slides were used to promote adhesion of the paraffin-section to the slides, which were then dried. The dried slides were de-paraffinized, and antigen retrieval was performed by automated antigen retrieval machine for 20 minutes in cell condition solution (Ethylenediaminetetraacetic acid pH 9.0). Sections were blocked for 1 h with 15–20% normal goat serum (Gibco Life Technologies, Grand Island, NY, USA), prior to incubation with primary antibody for 2 h at room temperature or overnight at 4 °C. Secondary rabbit antibodies were used to detect primary antibodies, followed by streptavidin-tagged horseradish peroxidase (Ventana Medical Systems, Tucson, USA). Diaminobenzidine (DAB, Sigma-Aldrich, St. Louis, Missouri, USA) was used to induce signalling, and bluing reagent (Ventana Medical Systems, Tucson, USA) was used as a counterstain. Images of IHC slides were visualized by optical microscopy (Leica, Wetzlar, Germany) and rendered using Leica software. For IHC, p-STAT3 (Tyr705) and NF-κB p65 antibodies were used.