Sc 654

The sections were prepared at 0.4 micron thickness and blocked with Ultra V block (LabVision, TA-125-UB^®). Deparaffinized sections were evaluated by IHC for the expression of intracellular adhesion strength molecule 1 (ICAM-1) and endothelial nitric oxide synthetase (eNOS). For this, the sections were incubated with 1/100 dilution of an ICAM-1 antibody (sc-107, Santa Cruz Biotechnology, CA^®, USA) for 60 min and 1/50 dilution of an eNOS antibody (sc-654, Santa Cruz Biotechnology, CA^®, USA) for 60 min.
The stain retention and intensity for the IHC experiments were graded as -, +, and ++ (-, no staining; +, slight staining; and ++, severe staining), with reference to the scoring system by Shapira et al.^{[9 (link)]} IHC findings were graded as follows: a score of 0 was assigned to (-) indicating no endothelial staining; score of 1 was assigned to (+) indicating faint staining across the entire endothelium, staining was seen at 10x zoom under a light microscope; while a score of 2 was assigned to (++) indicating marked staining across the entire endothelium, staining was seen even at 4x zoom under a light microscope. All pathological examinations were carried out by a pathologist blinded to the study design.

Western blots were performed to assess the renal cortex and medulla protein levels of eNOS and iNOS. Short isoform-specific primary antibodies to eNOS (1 : 1000 dilution, SC-654, Santa Cruz Biotech, Santa Cruz, CA) and iNOS (iNOS-A, 1 : 2000, Alpha Diagnostics International, San Antonio, TX) were used. Goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution, number 20320, Alpha Diagnostics International, San Antonio, TX) was subsequently applied. β-Actin was measured as an internal control using a monoclonal primary antibody (1 : 2000 dilution, A2228, Sigma-Aldrich Corporation, St. Louis, MO) and horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1 : 30 000 dilution, A9044, Sigma-Aldrich Corporation, St. Louis, MO). Membranes were detected with ECL immunoblotting detection reagents (GE Healthcare, Piscataway, NJ) and bands were quantified using a Chemi Doc chemiluminiscent detection system and Quantity One software (Bio-Rad, Richmond CA). The results were expressed as NOS/β-actin density ratio.