2.3. Immunohistochemical Study

Detection of protein localization was performed as previously described [11–13]. Paraffin-embedded kidney sections were cut to 4 μm in thickness. The slides were deparaffinized and endogenous peroxidase was blocked by treatment with 3% H₂O₂. The sections were incubated with the primary antibody to PKCβI (1 : 400; Santa Cruz Biotechnology), PKCβII (1 : 400; Abcam), NHE1 (1 : 100; Santa Cruz Biotechnology), or NHE3 (1 : 200; Santa Cruz Biotechnology) at 4°C overnight, followed by the respective horseradish peroxidase-linked secondary antibody (Bio-Rad), and then reacted with 3,3′-diaminobenzidine (DAB) solution (Sigma). As a negative control, the primary antibody was omitted, resulting in negative staining. In a blinded manner, three pathologists independently scored the staining intensity on a semiquantitative five-tiered grading scale from 0 to 4 (0 = negative; 1 = trace; 2 = weak; 3 = moderate; and 4 = strong) as previously described [11–13, 19].