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).