Immunohistochemical Analysis of Kidney and Liver

Sliced paraffin sections were immersed in xylene for deparaffinization, rehydrated to PBS^- from ethanol, and then heated in a microwave oven to 100°C for 15 min in 10 mM sodium citrate buffer (pH 6.0) to unmask antigens. The sections were incubated in 0.3% H₂O₂/methanol to destroy endogenous peroxidase activity, and blocked with 1% bovine serum albumin and 0.05% NaN₃ in PBS^- for 30 min at room temperature, and then incubated with a primary antibody overnight at 4°C [23 (link), 24 (link)]. The primary antibodies used for immunohistochemistry were anti-pERK1/2 (M8159, dilution 1:200; Sigma), anti-pS6 (#4858, dilution 1:100; Cell Signaling), anti-Ki67 (ab16667, dilution 1:100; Abcam). The sections were rinsed and incubated with a biotinylated anti-mouse and anti-rabbit IgG/IgA/IgM secondary antibody (Histofine; Nichirei Biosciences, Tokyo, Japan) for immunohistochemical staining. Immune reaction products were developed with 3,3′-diaminobenzidine. Cells with positive signals were counted in 10 random fields of either kidney or liver sections obtained from 6 rats in each group by a naive observer using a 40× objective. In the kidney, we counted positive cells in both normal and cystic structural areas.