Immunofluorescence staining
Kidney samples were mounted with O.C.T. compound medium (Tissue-Tek) and stored at -80 °C. Frozen samples were cut into 4-μm sections followed by immediate fixation (10% PBS-buffered formalin), washing, and dehydration. Sections were blocked with 10% PBS-buffered horse serum for 1 hour at room temperature, incubated with diluted primary antibodies at 4 °C overnight, and exposure to second antibodies and DAPI. Images were captured using A1R MP+ multiphoton confocal microscope (Nikon) and AxioCam HRc digital camera (Carl Zeiss).