This article describes a method for enhancing the transparency of fixed mouse kidney tissue to visualize sympathetic nerve distribution using light-sheet fluorescence microscopy. The process involves delipidation and decolorization using CUBIC-L, followed by immunostaining and refractive index matching with CUBIC-R+.
Take a fixed mouse kidney. Lipids in the tissue cause light scattering, and pigments cause light absorption, making the cells opaque.
Treat the tissue with CUBIC-L for delipidation and decolorization. Amino alcohol in CUBIC-L removes lipids and pigments, minimizing light scattering and increasing transparency.
Detergent molecules in CUBIC-L also permeabilize the cells.
Introduce a primary antibody cocktail that binds to cytoplasmic tyrosine hydroxylase in neurons of the sympathetic nerves and alpha-smooth muscle actin in vessel smooth muscle cells.
Add fluorophore-labeled secondary antibodies that bind to the primary antibodies.
Perform tissue postfixation to preserve the antibody treatment.
Immerse the tissue in CUBIC-R+ that replaces water and matches the refractive index of proteins, further minimizing light scattering and increasing transparency.
Under a light-sheet fluorescence microscope, light sheets illuminate the tissue, exciting the fluorophores.
Capture the fluorophore-emitted light and reconstruct a three-dimensional image, displaying the sympathetic nerve distribution around the arteries in the kidney.
To begin, perform the immersion fixation immediately after the perfusion fixation and kidney sampling by immersing the kidney in 4% paraformaldehyde at 4 degrees Celsius for 16 hours. Give three washes using PBS for two hours each. Perform the decolorization and the delipidation process by immersing the fixed kidney in seven milliliters 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 antibody 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.
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