Zli 9031

Immunohistochemistry was performed as described in a previous study (33 (link)). The paraffin embedded EC tissues and normal tissues from the same donors who met the requirements were collected. First, each tissue was fixed in 4% paraformaldehyde (PBS; Solarbio) for 48 h. Next, ~4 µm consecutive paraffin sections were cut, heated, dewaxed and dehydrated with immunohistochemical method in accordance with the protocol. EDTA solution of 1 mM (pH 8.9–9.1) was used for antigen retrieval and then endogenous peroxidase of tissues was removed. Then, sections were incubated in primary antibody dilution (antibody: PBS 1:500) against TRA2B (cat. no. sc-166829; dil, 1:500; Santa Cruz Biotechnology, Inc.) overnight at 4°C and washed by sterilized PBS at least 3 times. The tissues were then incubated in the presence of peroxidase-conjugated goat anti-rabbit antibody IgG which served as a secondary antibody (mouse, monoclonal, cat. no. A0286; dil, 1:1,000; Beyotime Institute of Biotechnology) at 37°C for 2 h. Then, the sections were stained with diaminobenzidine (ZLI-9031) and counterstained with hematoxylin solution (ZLI-9643) (both from ZSGB-Bio). Ten different fields per group were randomly selected and recorded under a light microscope (Olympus Corp.).

IHC and Western blot analyses were performed as described elsewhere^{10 (link),20 (link)}. For IHC, after primary antibody binding, peroxidase-conjugated goat anti-rabbit antibody IgG was bound as a secondary antibody. Tissue sections were then stained with diaminobenzidine (ZSGB-BIO, ZLI-9031), counterstained with hematoxylin (ZSGB-BIO, ZLI-9643), and examined under a light microscope (CKX41; Olympus Corp., Tokyo, Japan). Western blot band intensities were determined by densitometry analysis using the Image J software (National Institutes of Health, Bethesda, MD, USA), and the results were expressed either as the ratio of target protein to β-actin or as the ratio of the phosphorylated form of a protein to the total amount of that protein.