Kidney Histological Analysis Protocol

Histological analysis was performed as described previously^{43 (link),50 (link)}. The kidneys were fixed with 4% paraformaldehyde overnight and embedded in paraffin. Sections (4 μm thick) were stained with periodic acid–Schiff and Masson’s trichrome. Paraffin sections of kidney tissue were also stained with antibodies against F4/80 (rat monoclonal; Abcam, Japan) and type 3 collagen (rabbit polyclonal; Abcam, Japan), as described previously^{49 (link)}. Briefly, after 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 the anti-F4/80 antibody (diluted 1:100) and anti-type 3 collagen antibody (diluted 1:100) at room temperature for 2 hours. Morphometric analysis was performed using a BZ-9000 fluorescence microscope (Keyence, Japan).