Encyclopedia of Experiments: Immunology
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Transcript
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.
