Nbp2 13328

Immunohistochemical staining was performed on FNB-fixed, deparaffinized renal sections for NaPi-2a. Heat-induced epitope retrieval was performed by microwaving the sections (2x 5 min) in 0.1 M citrate buffer (pH 6.0). The tissue sections were then blocked with normal goat serum (20% in TSB with 1% Triton X-100) for 20 min and incubated overnight with polyclonal rabbit anti-human NaPi-2a (1:40; Novus Biologicals; NBP2-13328). Biotinylated goat anti-rabbit (Vector Laboratories) was used as secondary antibody. Avidin/biotinylated peroxidase complex (VECTASTAIN ABC KIT, Vector Laboratories) was added as signal amplifier and 3-Amino-9-ethylcarbazole (AEC, Sigma-Aldrich) was used as substrate. The sections were counterstained with hematoxylin. Sections in which the primary antibody was omitted were used as negative controls.

Protein was extracted from grounded kidney tissue in T-PER protein extraction reagent (Thermo Scientific, Rockford, IL) with Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL) using a Ultra-Turrax homogenizer. Protein concentration was determined by the BCA assay (Thermo Scientific, Rockford, IL). 30μg protein were run on Mini-Protean TGX stain-free precast gel (Bio-Rad, Germany) under reducing conditions and transferred onto nitrocellulose membranes (Bio-Rad, Germany). Membranes were blocked with 5% bovine serum albumin (BSA) (Roche, Germany) in phosphate buffered saline (PBS) with 0.05% tween. Primary and secondary antibodies were diluted in PBS with 3% BSA. The antibodies used were monoclonal anti-human Napi2a (1:1000) (NBP2-42216, Novusbio), polyclonal anti-human Napi2a (1:1000) (NBP2-13328, Novusbio), polyclonal anti-human Pit2 (1:500) (HPA026540, Atlas Antibodies), polyclonal anti-mouse klotho (1:2000) (ab154163, Abcam), polyclonal anti-human Park7 (1:1000000) (ab18257, Abcam). The secondary antibodies used were anti-rabbit (1:2000) (P0448, Dako) and anti-mouse (1:1000) (P0447, Dako). Western blot quantifications were performed with ImageJ.