Whole-Kidney Three-Dimensional Staining with CUBIC

This protocol allows a visualization of three-dimensional structures in full kidney, which can lead to innovative discoveries in kidney research by providing a macroscopic perspective. Whole-mount organ staining is challenging because of the difficulty in antibody penetration. This protocol has demonstrated the high quality, whole-mount staining of the kidneys by the antibodies.

To begin, perform the immersion fixation immediately after the perfusion fixation and kidney sampling, by immersing the kidney in 4%paraformaldehyde at four degrees Celsius for 16 hours. Give three washes using PBS for two hours each. Perform the decolorization and delipidation process by immersing the fixed kidney in seven millimeters of 50%CUBIC-L in a 14 milliliter round-bottom tube, with gentle shaking at room temperature.

After six hours, immerse the kidney in seven milliliters of CUBIC-L in a 14 millimeter round-bottom tube, with gentle shaking at 37 degrees Celsius for five days. Refresh CUBIC-L every day. Once the decolorization and delipidation are complete, use a dispensing spoon for sample handling and wash the kidney thrice with PBS at room temperature for two hours.

Immunostain the delipidated kidney in primary antibodies diluted in staining buffer solution, with gentle shaking in an incubator shaker at 37 degrees Celsius for seven days. Then, wash the kidney with 0.5%Triton X-100 in PBS, also known as PBST, at room temperature for one day. For secondary antibodies staining, treat the kidney with secondary antibodies in the staining buffer, in the dark, with gentle shaking at 37 degrees Celsius for seven days.

Fix the staining with a PBST wash at room temperature for one day. Post-fixation, immerse the kidney in 1%formaldehyde in PB for three hours, and wash it with PBS at room temperature for six hours. Perform the refractive index matching by immersing the kidney in seven milliliters of 50%CUBIC-R+in a 14 milliliter round-bottom tube with shaking for one day, followed by treatment with seven milliliters of CUBIC-R+with shaking at room temperature for two days.

With the help of the immune staining method, sympathetic nerves and arteries in a whole kidney were visualized in 3D kidney imaging. Moreover, abnormal renal sympathetic nerves were visualized after ischemia-reperfusion injury. This protocol also facilitated visualizing sympathetic nerves in the whole spleen.

The data quantification process for the three-dimensional immunofluorescent staining is displayed here. In addition, collecting ducts, the S1 segment of proximal tubules, and the glomeruli were successfully visualized in a whole kidney. The suppressed glomerulomegaly, resulting from drug administration in the early stages of diabetic kidney disease, was also visualized in 3D imaging.

This protocol can label the specific molecule within an entire kidney. Thus, researchers can analyze structure alteration in the vasculature or find rare events in kidney tissues.