Masson s trichrome staining kit

The sections were removed from the embedding agent, placed in xylene, and then rehydrated with decreasing concentrations of ethanol. For H&E staining, the sections were placed in a haematoxylin dye solution for 8–10 min and then stained with an eosin solution for 8–10 min after differentiation with differentiation solution (1% HCl and 70% alcohol). A Masson’s trichrome staining kit (Sigma, USA) was used, and staining was performed according to the manufacturer’s protocol. For immunohistochemistry, after deparaffinization and hydration, the sections were treated with H₂O₂ for 30 min to block endogenous peroxidase activity and treated with trypsin and pepsin to retrieve the antigens. Next, the sections were blocked with 3% BSA for 30 min and then incubated with the primary antibody at 4 °C overnight. Afterwards, the sections were incubated with a secondary antibody (HRP-labelled) and finally visualized with DAB through a chromogenic reaction. After nuclear counterstaining, dehydration and mounting, the stained sections were observed and photographed under a light microscope (Leica, Germany).

Tissues were fixed with 4% formalin overnight, embedded in paraffin and cut into 6 μm slices. H&E and Masson staining (Masson’s trichrome staining kit, Sigma) were performed separately on consecutive tissue sections, and images were obtained using a microscope (Leica DM4000, Wetzlar, Germany). Quantification of fibrosis was conducted using ImageJ (NIH) as the percentage of blue collagen-stained area relative to the total tissue in one field.