JoVE Encyclopedia of Experiments
Microbiology
0 views • 2:27 min • August 31st, 2026
Begin with frozen recellularized rat liver tissue, containing human hepatocytes, embedded in cryomedium.
In this tissue, the transduced hepatocytes express the emerald green fluorescent protein (EmGFP), a reporter protein encoded by the viral vector.
Use a cryotome to cut thin sections that retain cellular and tissue architecture. Transfer the sections to a slide.
Treat the sections with a mild detergent to permeabilize the cell membrane.
Apply an anti-EmGFP primary antibody that specifically binds to the fluorescent reporter protein expressed in transduced cells.
Wash to remove unbound antibodies.
Introduce a secondary antibody conjugated to a red fluorophore, which binds to the primary antibody, and then wash.
Add a DNA-specific blue fluorescent dye that binds to cellular DNA.
Under a fluorescence microscope, the colocalization of red fluorescence with blue-stained nuclei confirms that the transduced hepatocytes in the liver tissue successfully express the viral reporter protein.
Using a scalpel, slice samples from each liver lobe that are about half a centimeter thick and 1.5 to 2 centimeters long. Incubate the slices in 4% paraformaldehyde with 4%sucrose for 90 minutes at four degrees C
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