Lu-Fc Binding Assay on Mouse Kidney Sections

The Lu-Fc binding assay was performed as described previously [19 (link)]. Briefly, Lu-Fc was diluted with PBS(-) containing 10 μg/ml of antibody or 5–12 × 10¹¹ pfu/ml of phage antibodies and was then placed on adult mouse kidney sections. The α5-containing laminin, the specific ligand of Lu, is enriched in the basement membrane of the kidney. Therefore, we could conveniently perform the binding assay for Lu on the tissue without purifying the α5-containing laminins. Animal studies were approved by the Animal Research Committee of Tokyo University of Pharmacy and Life Sciences (P15-10). Mice were sacrificed under deep anesthesia with Pentobarbital. Bound Lu-Fc was detected with anti-human IgG antibody conjugated to Alexa488 (ThermoFisher Scientific). The anti-laminin α5 polyclonal antibody was used for counter staining. The fluorescence intensities of the bound recombinant proteins were normalized with those of laminin α5 staining in the same areas and quantified using a BZ-analyzer (Keyence, Osaka, Japan).