2.5. Immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses

Formalin-fixed kidney samples were processed and 5-µm thick paraffin sections were used for immunohistochemistry (IHC). In brief, deparaffinized kidney sections were subjected to 3% hydrogen peroxide followed by antigen retrieval. The sections were blocked with 2% (w/v) non-fat skim milk solution, and incubated with the primary antibody against myeloperoxidase, oxidized proteins, nitrated proteins detected by anti-3-NT antibody, or collagen type 1A1 at 4 °C overnight. After subsequent washing steps to remove the unbound antibody, the attached primary antibody was then linked to the dextran polymer per manufacturer's protocol (Envision kit, Dako, Carpinteria, CA, USA). The final reaction was performed by immersing the sections in a solution of 3,3′-diaminobenzidine (DAB). The sections were then counterstained with hematoxylin. For detection of DNA strand breaks to characterize apoptotic cell death in kidney tissues, the ApopTag Peroxidase in situ apoptosis detection kit (cat. # S7100) (Millipore, Billerica, MA) was used. TUNEL-positive kidney cells were counted in 10 high-power microscope fields for all four groups and tabulated.