Immunohistochemical and Immunofluorescence Staining of Kidney Sections

The IHC and IF staining was performed according to previously published protocols [13 (link)–15 (link)]. For IHC and IF staining, rehydrated paraffin sections were treated with 3% H₂O₂ for 30 minutes and then incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 minutes at room temperature. Then, the sections were incubated with the following primary antibodies: zonula occludens-1 (ZO-1; 1:300, Abcam, Cambridge, MA, USA), Wilm's tumor suppressor gene 1 (WT-1; 1:1000, Abcam, Cambridge, MA, USA), kidney injury molecule (KIM-1;1:200, R&D systems, Minneapolis, MN, USA), fibroblast specific protein (FSP-1; 1:200, Abcam, Cambridge, MA, USA), P-cadherin (1:100, Novus, Littleton, CO, USA), podocin (1:100,Sigma, St. Louis, Mo, USA), dystrophin (1:100, Abcam, Cambridge, MA, USA), fibronectin (1:200, Abcam, Cambridge, MA, USA), dystroglycan (1:50, Abcam, Cambridge, MA, USA), synaptopodin (1:100, Santa Cruz, CA, USA), CD14 (1:50, Santa Cruz, CA, USA), CD68 (1:100, Abcam, Cambridge, MA, USA); sections incubated with irrelevant antibodies served as controls. Three kidney sections were analyzed from each rat. For quantification, three randomly selected high power fields (HPFs; 200× for IHC; 400× for IF) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was determined by summation of all numbers divided by 9.