Endogenous peroxidase blocker

Briefly, the samples were fixed with 4% formaldehyde for 48 h at room temperature and embedded in paraffin. Then, the paraffin-embedded tissue sections at 5-8 μm thickness were transferred onto glass slides. The slides were deparaffinized, rehydrated, and pressure cooker heated for 2-5 min in EDTA for antigen retrieval. Endogenous peroxidase activity was inactivated by adding an endogenous peroxidase blocker (OriGene, Beijing, China) for 15 min at room temperature. Slides were incubated overnight at 4°C with KLF5 (Proteintech, 66850-1-Ig), EphA2 (Santa Cruz, sc-398832), and EphA2 pS897 (Cell Signaling, #6347) antibodies. Next, the slides were washed three times with 1×PBS and incubated with secondary antibody (OriGene, Beijing, China) at room temperature for 20 min, DAB concentrate chromogenic solution (1:200 dilution of concentrated DAB chromogenic solution), counterstained with 0.5% hematoxylin, dehydrated with graded concentrations of ethanol for 3 min each (70%-95%-100%), and finally stained with dimethyl benzene. Immunostained slides were evaluated by light microscopy. IOD score: integrated optical density score.

Paraffin-embedded clinical TNBC specimens were obtained from the Department of Pathology, Henan Provincial People’s Hospital, Zhengzhou University, Henan, China. Two tissue microarrays containing 135 TNBC breast cancer tissues were constructed. For the IHC assay, the slides were deparaffinized, rehydrated, and pressure cooker heated for 2.5 min in EDTA for antigen retrieval. Endogenous peroxidase activity was inactivated by adding an endogenous peroxidase blocker (OriGene, China) for 15 min at room temperature. Slides were incubated overnight at 4°C with anti-TNFAIP2 (1:200) or anti-ITGB4 (1:500). After 12 hr, the slides were washed three times with PBS and incubated with secondary antibodies hypersensitive enzyme-labeled goat anti-mouse/rabbit IgG polymer (OriGene, China) at room temperature for 20 min, DAB concentrate chromogenic solution (1:200dilution of concentrated DAB chromogenic solution), counterstained with 0.5% hematoxylin, dehydrated with graded concentrations of ethanol for 3 min each (70–80–90–100%), and finally stained with dimethyl benzene immunostained slides were evaluated by light microscopy. The IHC signal was scored using the ‘Allred Score’ method.