Histological and Immunohistochemical Analysis of Kidney Tissues

Kidneys were cut transversely and fixed in 10% neutral-buffered formalin, embedded in paraffin, and treated with periodic acid–Schiff (PAS) and elastica Masson trichrome stains. Deparaffinized sections were prepared by routine procedure. Immunohistochemical staining of formalin-fixed, paraffin-embedded kidneys was performed after antigen retrieval in Target Retrieval Solution (Dako, Glostrup, Denmark). Abs against the following proteins were used: uromodulin (UMOD; AbD Serotec, Raleigh, NC, USA), Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2; StressMarq Biosciences, Victoria, BC, Canada), Na⁺-K⁺-ATPase (Abcam, Cambridge, United Kingdom), F4/80 (AbD Serotec), Na⁺-Cl⁻ cotransporter (NCC; EMD Millipore, Billerica, MA, USA), aquaporin-2 (AQP2; Abcam), and malondialdehyde (JaICA). All slides were counterstained with hematoxylin. We performed Gb3 staining with Shiga toxin 1 B subunit and transmission electron microscopy as described in Taguchi et al. (20 (link)). Toluidine blue staining was performed with 0.05% toluidine blue solution. We used an all-in-one fluorescence microscope (BZ-X700; Keyence, Osaka, Japan) to document staining. Information on Abs used in this study is provided in Supplemental Table 1.