Confirming Viral Transduction in Recellularized Rat Liver Using Immunohistochemical Staining

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 Celsius.

Then wash the samples three times with PBS for one minute per wash. Follow the washes by incubating the samples overnight in 8%sucrose at four degrees Celsius. The next day, load the samples into cryomolds containing some fixing medium.

Avoid introducing any air bubbles. Once loaded, fully cover the samples with fixing medium and put them at negative 80 degrees Celsius. Once frozen, prepare 10 micron cryosections from the samples using a cryotome.

Stain the samples as needed to confirm the recellularization.