Histological and Immunohistochemical Analysis of Kidney Tissue

Kidney tissue samples were fixed, dehydrated, and set into paraffin before being sliced at 4 µm and added to slides stained with hematoxylin and eosin (H&E). The samples were examined and photographed for histopathological analysis using a Nikon Eclipse 80i microscope and a Canon SX620 H 20 megapixel digital camera (Awad et al., 2018 (link)). Immunohistochemical analysis was performed by embedding the slices in paraffin, rehydrating these, then soaking in H₂O₂ (2%) for 15 m, then inhibiting the peroxidase activity using PBS. A 5% bovine serum albumin was used to block nonspecific binding sites. Dilutions of 1:500 Bcl‐2‐associated X protein (Bax) Antibody (Catalog # sc‐23959), cyclooxygenase‐2 (Cox‐2) antibody (Catalog # sc‐19999), and polyclonal antibodies (Santa Cruz Biotechnology) were added to the slides and incubated at 4°C overnight. After washing the slides three times with PBS, a biotin‐conjugated secondary antibody (1:2000 dilution, cat# sc‐2040) was added. After developing the reaction with 3,3‐diaminobezidine tetrahydrochloride, the slides were counterstained using hematoxylin and the number of positively stained cells was compared to the total number of cells to determine how many of those cells were immunoreactive for Bax and Cox 2 (Nassan et al., 2018 (link)). Significance was determined using ANOVA for three different samples per group.