Growing interest in studying large, multi-cellular structures led to the development of this optic clearing technique which allows for a three-dimensional visualization and quantification of large structures within transparent organs. This protocol provides a simple, cheap, easy to set up and comprehensive tool to investigate tissue in three dimensions. Multi-layered nature of a disease cannot always be simulated by traditional two-dimensional techniques.
Three-dimensional analysis of tissue can help to better diagnose and monitor disease. This method is not restricted to kidney tissue and can be applied to any normal and diseased tissue. This simple protocol is easy to follow for any scientist with some experience in animal handling.
It is important to test different antibodies in case of poor antibody labeling, and conduct secondary antibody only controls. Start by preparing heparin containing PBS and 3.0%paraformaldehyde in 1X PBS according to the manuscript directions and transferring the solutions into separate 50 mL plastic syringes. Paraformaldehyde is toxic and should be prepared in the fume hood.
Collect and profuse the kidney according to the manuscript directions. Then, remove the capsule and cut the kidney into one mm thick coronal slices. Immerse the kidney slices with the prepared paraformaldehyde solution in a 15 mL conical tube.
After fixation, wash the kidney slice twice with 1X wash buffer for one hour on a horizontal rocker and then proceed with antigen retrieval. Heat up 300 mL of 1X antigen unmasking solution in a 500 mL beaker. Then, enclose a kidney slice in an embedding cassette permeable to the heated buffer and stir it for one hour at 92 to 98 degrees Celsius.
It is important to keep the temperature stable between 92 and 98 degrees Celsius for this antigen retrieval step. Then transfer the slice into 10 mL of 1X wash buffer with 0.1%Triton X-100 and rock it overnight. The next day, wash the slice twice with 10 mL of fresh 1X wash buffer for one hour.
Dilute the primary antibody in 500 microliters of normal antibody diluent. Gently rock the kidney slice in diluted primary antibody for four days at 37 degrees Celsius. After the incubation, wash the kidney slice in 10 mL of 1X wash buffer overnight at room temperature, changing the buffer once after eight hours.
Dilute the secondary antibodies in 500 microliters of normal antibody diluent and incubate with the kidney slice for four days at 37 degrees Celsius. Please note that from this step forward, the kidney slice should be protected from light. After incubation with secondary antibodies, wash the kidney slice in 10 mL of 1X wash buffer overnight at room temperature.
To dehydrate the kidney slice, transfer it to five mL of high grade 100%ethanol and rock it for two hours at room temperature, refreshing the ethanol after one hour. Immerse the kidney slice in two mL of ethyl cinnamate and rock gently overnight. When ready to image the tissue, add 600 to 1000 microliters of ethyl cinnamate into a glass-bottom dish and transfer the kidney slice into the dish.
Place a round cover slip on the kidney slice and seal the dish with paraffin film. Place the dish onto the microscope imaging platform and image the tissue according to the manuscript directions. This protocol has been used to successfully perform 3D analysis on kidney tissue and evaluate cell functions and interactions.
The method may cause fluorescent reporter quenching but other strong fluorescent proteins may resist signal quenching. Abdominal aortic profusion can improve antibody diffusion. In some instances, poor antibody penetration results in a superficial signal.
This can be corrected with higher concentrations or replenishment of antibody. Intravascular delivery should be considered if the antigen of interest is expressed in kidney vasculature. However, antibodies targeting proteins in the apical membrane of tubule epithelial cells do not cross the glomerular filtration barrier and cause unspecific signals.
The tissue can be labeled with the antibodies to detect a specific cell population or to visualize whole tubule segments. Furthermore, colocalization of proteins in 3D can be achieved by combining multiple antibodies. It is important to remember that the antibody labeling can be improved with good kidney profusion, the use of antigen retrieval step, and high antibody concentration.
Some strong reporter proteins may resist quenching of fluorescent signal following this protocol. To test this, skip the antigen retrieval and antibody labeling steps. This technique respects the three-dimensional nature of structures and opens up many new areas for research.
It eliminates a required assumptions and interferences associated with traditional two-dimensional analysis.
