Nb110 96417

Histological analysis was performed as described previously.14, 15 Renal tissue from mice was fixed with 4% paraformaldehyde and subsequently embedded in paraffin. Sections (4‐μm thick) were stained with periodic acid‐Schiff and Masson's trichrome. Immunohistochemistry was performed as described previously.13, 14, 18 Sections of 4‐μm thickness were dewaxed and rehydrated, and antigen retrieval was performed by microwave heating. The sections were blocked for endogenous biotin activity using peroxidase blocking reagent (DAKO) and treated for 60 minutes with 10% normal goat serum in phosphate‐buffered saline. The sections were then incubated with one of the following: anti‐ATRAP antibody (diluted at 1:100), anti–calbindin D‐28K antibody (C9848 [Sigma–Aldrich]; diluted at 1:100), or anti–megalin antibody (NB110‐96417 [Novus Biologicals]; diluted at 1:100). The characterization and specificity of the anti–mouse ATRAP antibody was described previously in detail.14, 18, 20

Immunohistochemistry was performed as described previously.^{10 (link), 20 , 52 , 53} The sections were incubated with one of the following: (1) anti-ATRAP antibody diluted to 1:100, (2) anti-aquaporin 2 antibody (1:200; 254-271, CALBIOCHEM, Darmstadt, Germany), (3) anti-calbindin D-28K antibody (1:3000; C9848, Sigma-Aldrich, St Louis, MO), or (4) anti-megalin antibody (1:1000; NB110-96417, Novus Biologicals, Littleton, CO).