Hydrogen peroxide

IHC was performed using standard techniques. Briefly, 5-µm paraffin-embedded TMA sections were dewaxed using xylene (Sinopharm, China)and rehydrated in graded alcohols (Sinopharm, China). Next, blocked endogenous peroxidase with 3% hydrogen peroxide (sinopharm, China). Antigen retrieval was accomplished via adding 10 mM citrate buffer (pH 6.0) (Sinopharm, China) to the TMA sections and putting them into a high-pressure cooker. Then, the TMA sections were incubated with 1% bovine serum albumin (BSA; Sigma, German) for 20 min to reduce nonspecific protein binding. The recombinant rabbit monoclonal CDC45 antibody (1:50; HuaBio, Hangzhou, China) were next used to treat the TMA sections at room temperature for 1 h. They were then rinsed with phosphate-buffered saline (PBS; HuaBio, Hangzhou, China) and incubated with biotinylated secondary antibody (MXB, Fuzhou, China) for 30 min at room temperature. Subsequently, the TMA sections were stained with DAB chromogen (Gene Tech, Shanghai, China), counterstained with Mayer’s haematoxylin (HuaBio, Hangzhou, China), dehydrated with gradient alcohol and xylene, and mounted on slides. Finally, the TMA sections were viewed under a Nikon light microscope.

Detection of the relative expression of vimentin in fibroblasts was performed as previously described (26 (link)), using the Vimentin immunohistochemistry kit (Nanjing KeyGEN Biotech Co., Ltd, Jiangsu, China) according to the manufacturer’s instructions. Cells of passage three were seeded at a density of 3.0×10⁵ cells/ml into a six-well plate containing sterilized coverslips. Following two days of incubation, cells were fixed for 30 min with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd), washed three times with PBS and then incubated in 3% hydrogen peroxide (Sinopharm Chemical Reagent Co., Ltd) at room temperature for 5 min. Following blocking with goat serum (Nanjing KeyGEN Biotech Co., Ltd) for 30 min, samples were incubated with mouse anti-human monoclonal anti-vimentin antibodies (1:100; Nanjing KeyGEN Biotech Co., Ltd) overnight at 4°C followed by incubation with biotin-labeled goat anti-mouse immunoglobulin G (1:100 in antibody diluents; Nanjing KeyGEN Biotech Co., Ltd) at 37°C for 30 min. Samples were then incubated with horseradish peroxidase-labeled streptavidin solutions (Nanjing KeyGEN Biotech Co., Ltd) at 37°C for 30 min and the signals were evaluated following the addition of diaminobenzidine (Nanjing KeyGEN Biotech Co., Ltd) and hematoxylin staining under an inverted microscope (BX53; Olympus).

Tissue sections (4 μm) were prepared for immunohistochemical study. The sections were processed with deparaffinization, retrieval in EDTA solution (Aspen, Wuhan, China), and incubation in 3% hydrogen peroxide (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). The samples were incubated overnight at 4°C with primary antibody of rabbit polyclonal anti-Ki67(Affinity Biosciences, Cincinnati, USA), then incubated with the HRP-labeled goat anti-rabbit second antibody (Aspen, Wuhan, China) at 37°C for 50 min. Brown-stained nuclei in cells were considered positive and the Ki67-positive rate was quantified by ImageJ software.

For immunohistochemistry, the paraffin slices were subjected to dewaxing and hydration firstly. After that, the slides were put into antigen retrieval solution and heated for 10 min to retrieve the antigen. The slides were blocked with 1% BSA (Sangon biotech, China) for 15 min at room temperature after treatment with 3% hydrogen peroxide (Sinopharm, China) for 15 min. Then the p-JAK2 (AP0531, 1:50, ABclonal Technology Co., Ltd., China) and p-STAT3 (AP0705, 1:50, ABclonal Technology Co., Ltd., China) primary antibodies were added to the slides for incubation overnight at 4 °C. The HRP labeled goat anti-rabbit IgG (D110058, 1:200, Sangon biotech, China) secondary antibody was used for incubation for 60 min at room temperature. DAB chromogenic solution (Beijing Solarbio Science & Technology Co.,Ltd, China) was used to develop the color, and the sections were counterstained with hematoxylin (Beijing Solarbio Science & Technology Co.,Ltd, China). Finally, the slices were observed under the microscope (BX53, Olympus Corporation, Japan) and photographed.