Ab94764

Because the surface of MNPs is composed of PEG, we performed florescent IHC for PEG to detect renal proximal tubular localization of MNPs administered. Kidneys from mice treated with 38 mg/kg NBP MNP 6 hours before renal IR injury were fixed with 4% paraformaldehyde, dehydrated with 30% sucrose, frozen in OCT (Tissue-Tek), and cryosectioned (5 μm). Kidney sections were permeabilized with 0.2% Triton X-100 for 10 minutes and the sections were then autoclaved in 10 mM sodium citrate, pH 6.0, for 10 minutes to retrieve antigens. After blocking sections with PBS containing 10% normal rabbit serum, sections were incubated with 1:100 anti-PEG antibody (ab94764, Abcam) specific to the PEG backbone plus PHA lectin antibody (proximal tubule–specific marker, Molecular Probes) or plus aquaporin-2 antibody (collecting duct–specific marker, AQP-002, Alomone Labs). After incubating sections with specific fluorescent secondary antibodies (Thermo Fisher Scientific), kidney slides were counterstained with DAPI to visualize cell nuclei, mounted with Vectashield (Vector) mounting media, and imaged with a fluorescent microscope (Olympus IX81). We also performed PEG florescent IHC in the lung, spleen, and liver.