Immunohistochemical Analysis of Liver and Kidney Tissues

Paraffin-embedded sections of the liver and kidney tissues were de-paraffinized and rehydrated using graded alcohol concentration [25 (link)]. Antigen retrieval was achieved by boiling in 10 mmol/L sodium citrate buffer for 10 min and then steadily cooling to room temperature. Subsequently, the sections were blocked using 3% H₂O₂ in methanol for 15 min to inhibit endogenous peroxidase activity. Following washing in phosphate buffer saline (PBS), the sections were incubated overnight at 4 °C with monoclonal rabbit anti-p53 and anti-CD43 primary antibodies (Elabscience, Wuhan, China) at a dilution of 1:40. The sections were incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) at the same dilution of 1:500 for 2 h at 37 °C. The sections were washed in PBS, developed in prepared DAB chromogen solution, lightly counterstained with hematoxylin, dehydrated, mounted, and visualized under the light microscope (Leica DM2700 M, Wetzlar, Germany). The degree of positivity of the proteins (CD43 and p53) in the hepatic and renal cytoplasm was quantified using Image J software [26 (link)]. The photomicrograph is a representative slide from five animals, from which 3 slides were quantified.