Pv 8000

The paraffin sections of four 8-week-old APC^min/+ mice and human CRC tissue array (containing 74 normal tissues and 100 cancer tissues) were dewaxed with xylene and rehydrated through an ethanol gradient. Then, the sections were subjected to antigen retrieval with EDTA solution at 100 °C for 3 min in a pressure cooker, deactivation of endogenous peroxidase with 0.3% hydrogen peroxide, and blocking with 5% BSA. The paraffin sections were incubated with the ADCK1 antibody (HPA051012) overnight at 4 °C using an immunohistochemical kit (ZSGB-BIO, PV-8000), washed with PBST three times, and incubated with the secondary antibody at room temperature for two hours. Later, DAB (ZSGB-BIO, ZLI-9019) was used for color development for 2 min. After color development, the paraffin sections were stained with hematoxylin to identify cell nuclei. The paraffin sections were differentiated for 6 s with 1% hydrochloric acid/ethanol, and the liquid turned blue. The immunohistochemical score was evaluated with a Vectra 2.0 system to obtain the H value. The expression of the target gene in tumor tissues and normal tissues was evaluated based on the H values.

30 clinical samples were collected from Shanghai Chest Hospital after the informed consent was obtained was obtained from all subject. This study was approved by the ethical committee of the Shanghai Chest Hospital. The clinical information was listed in Table S1. Tissues were embedded in paraffin and cut into 5 µm sections. Dewaxing and rehydration were performed by placing slides in a decreasing gradient series of xylol and ethanol. Antigen recovery was performed by incubating the sections in a pH 6.0 citrate sodium solution at 100 °C for 20 min. After blocking endogenous peroxidase activity using a kit (ZSGB-BIO, PV-8000), the tissues were incubated with primary antibody (anti-DSTYK, 1;100, Proteintech, 20102-1-AP; anti-β-catenin, 1;100, CST, 8480; or anti-LDHA, 1;100, Proteintech, 19987-1-AP) at 4 °C overnight. Then, secondary antibody was added, and the tissues were incubated at room temperature for 1 h. The signals were developed using DAB (ZSGB-Bio, Beijing, China), and the sections were stained with hematoxylin. Tissues were examined under a microscope, and scoring was performed as described.

For histological staining, the samples were fixed, decalcified and embedded. The sections were cut at the thickness of 5 μm. The sections were stained with Hematoxylin & Eosin (H & E) or safranin O/fast green staining (SO). For immunohistochemistry analysis, goat two-step detection kit was used to detect the antigens according to the manufacturer’s instructions (ZSGB-BIO, PV-8000). The sections were incubated with 100 μl diluted primary antibody of rabbit polyclonal anti-NF-κB (#: 8242, 1:200, Cell Signaling Technology) or rabbit polyclonal anti-HIF-2α (#: A01248-1, 1:50, Boster) overnight at 4°C respectively, and then incubated with HRP-conjugated secondary antibody for 1 h at room. The reaction was visualized by incubating the sections with a DAB kit (#: SP-9000, ZSGB-BIO) and hematoxylin for 5 min at room temperature. The photos were taken by an inverted phase contrast microscope (Olympus CKX41-A32PH). The adapted histological parameters originated from the International Cartilage Repair Society (ICRS) II include: (1) matrix staining; (2) subchondral bone; (3) overall assessment. The defect surrounded by the dashed lines was defined as region of interest and employed for analysis. Three blinded readers graded the cartilage sections according to ICRS II parameters and criteria [19 (link)].