Histofine simple stain max po mouse kit

Serial sections were cut at 5 μm thickness and stained with hematoxylin and eosin (H&E). Histological analysis was performed according to the previously reported criteria [18 (link)]. After dewaxing and hydration, sections were incubated with an antibody against β-catenin (dilution 1:200; clone 14, BD Biosciences, Franklin Lakes, NJ, USA), a peroxidase-conjugated secondary antibody [Histofine Simple Stain MAX PO (Mouse) kit, Nichirei, Tokyo, Japan], reacted with 3,3′-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin.

The tubulointerstitial lesions and fibrosis were evaluated in renal tissue sections (5 μm) with Masson trichrome staining. Ten fields from the cortical areas were selected randomly per mouse. Tubulointerstitial lesions were defined as the accumulation of tubular dilatation/atrophy, tubule vacuole and cast formation, interstitial edema, and epithelial cell necrosis^{47 (link)}. Tubulointerstitial fibrosis was defined as the accumulation of the extracellular matrix (stained blue). The extent of tubulointerstitial injury and fibrosis was defined as a ratio relative to the entire cortical area with ImageJ software (National Institutes of Health, Bethesda, MD).
Immunohistochemical staining was performed as described previously^{47 (link)}. Deparaffinized kidney sections were heated for 20 min at 121 °C in a 10-mM citric acid solution for antigen retrieval and then incubated with antibody against F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA). The primary antibody was detected using the Histofine Simple Stain MAX-PO (mouse) kit (Nichirei, Tokyo, Japan) and peroxidase stain DAB kit (Nacalai Tesque, Kyoto, Japan). The nuclei were stained with hematoxylin.