Processing of Lentivirus-Transduced Mouse Intestinal Organoids for Immunohistochemical Analysis

Take a multi-well plate containing mouse intestinal organoids embedded in a gel matrix.
The organoid cells are transduced with a reporter gene using lentivirus. This gene expresses a reporter protein.
Add fixative to the well to preserve organoid tissue morphology.
Wash with buffer to remove residual fixative.
Resuspend the organoids in buffer, transfer them to a glass vial, and allow them to settle.
Replace the buffer with ethanol containing eosin dye, which stains the organoids to enable visualization.
Treat the organoids with increasing concentrations of ethanol to dehydrate them.
Add xylene to increase tissue transparency.
Decant the xylene, add molten paraffin, and incubate at high temperature to infiltrate the tissue.
Replace with fresh paraffin, ensuring complete infiltration.
Transfer organoids into a mold containing paraffin, center them, and allow the paraffin to solidify.
Add additional paraffin and insert an embedding cassette.
Paraffin-embedded lentivirus-transduced organoids are ready for immunohistochemical analysis.

Remove the medium from a single well with full-grown organoids, leaving the embedded organoids intact, and add one milliliter of 4% paraformaldehyde in PBS directly to the well. Replace the paraformaldehyde with one milliliter of PBS.

Resuspend the fixed organoids in the PBS and transfer to a glass vial. Let the organoids sink to the bottom for one minute. Next, pipette off the PBS and replace with 70% ethanol in which a couple of droplets of eosin solution are dissolved to enable visualization of organoids throughout the embedding process. Leave the organoids in 70% ethanol at room temperature for 30 minutes. Remove the 70% ethanol by carefully pipetting. The organoids can be visualized by eye because of the pinkish eosin color.

Replace the embedding solution with 96% ethanol and leave at room temperature for 30 minutes. In this manner, subsequently, pass the organoids through 70% ethanol, 90% ethanol, 96% ethanol, 100% ethanol, 100% ethanol, xylene, and xylene. Decant the last xylene wash and pour paraffin into the glass vial.

Put the vial immediately in the prewarm aluminum block at 70 degrees Celsius for 30 minutes. After 30 minutes, replace the paraffin with new clean paraffin with a prewarm Pasteur pipette. Pour the paraffin off and use a prewarm Pasteur pipette with a large opening to pipette the organoids into the paraffin block mold into a layer of liquid paraffin.

Warm a dissection needle with a Bunsen burner and manipulate all organoids as much as possible toward the center of the paraffin block mold. When localization of the organoids in the mold is satisfactory, chill the mold slightly to solidify the paraffin layer. Finish the block by pouring more paraffin on top and add a standard histological embedding cassette.