Aqp 002

The antibodies used included those against LC3B for western blotting (Cat. No. L7543, Sigma-Aldrich Inc.) and for immunohistochemistry (Cat. No. 0231-100/LC3-5F10, Nanotools), SQSTM1 (Cat. No. GP62-C, Progen), RAB9 (Cat. No. ab2810, Abcam), pS261-AQP2 (Cat. No. ab72383, Abcam), pS256-AQP2 (Cat. No. ab109926, Abcam & kindly provided by Prof. Tae-Hwan Kwon, Kyungpook National University, Korea), and total AQP2 (Cat No. AQP-002, Alomone).

Following bioreactor perfusion, kidneys were fixed in 10% neutral buffered formalin (NBF), imbedded in paraffin wax, and sectioned for immunochemistry (IHC). Primary antibodies for IHC were LTL (Vector labs, B1325, Newark, CA, USA), E cadherin (R&D, AF748, Santa Clara, CA, USA), Nephrin (R&D System, AF4269, Toronto, ON, Canada), KSP (Novus Biologicals, NBP1-59248, Littleton, CO, USA), AQP2 (Alomone Labs, AQP-002, Jerusalem, Israel). The primary antibodies were diluted in 0.1%FBS/0.1%Triton/PBS at a 1:100 ratio (except for E cadherin which was diluted at a 1:50 ratio). The secondary antibodies used were: Strepavidin 488 (Invitrogen, S32354, Waltham, MA, USA), Donkey anti-goat IgG Cy5 (Abcam, ab6566, Cambridge, UK), Donkey anti-sheep IgG NL557 conjugated antibody (R&D Systems, NL010), Donkey anti-rabbit IgG594 (Invitrogen, A21207), Donkey anti-rabbit IgG488 (Invitrogen, A21206). They were diluted in the same solution as the primary antibodies at a 1:500 ratio. Cell nuclei were stained with DAPI. Hematoxylin and Eosin (H&E) staining was also done to visualize tissue morphology.
Kidney was also stained with Periodic-acid Schiff (PAS) reagent staining. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was done as previously described [12 (link)].

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.