Characterization of Small Bowel Neuroendocrine Tumor Spheroids Using Immunofluorescence

Take small bowel neuroendocrine tumor spheroids in a tube.

Add a fixative solution to preserve the cells. 

To wash, centrifuge, and replace the fixative with buffer.

Centrifuge and discard the supernatant to remove any remaining fixative.

Add a buffer containing detergent and a blocking agent to permeabilize the cellular membranes and prevent non-specific antibody binding.

Wash with buffer to remove excess detergent and blocking agent.

Incubate with primary antibodies, which bind to the target protein specific to the neuroendocrine cell.

Wash with buffer to remove unbound antibodies.

Add fluorophore-tagged secondary antibodies that bind to the primary antibodies.

Rinse with buffer to remove excess antibodies.

Add a mounting medium containing a dye to stain the nucleus.

Transfer the spheroids onto a slide and mount with a cover slip.

Use a Fluorescence microscope to visualize neuroendocrine cells of the spheroid expressing specific proteins.

Fix the organoids by adding 500 microliters of paraformaldehyde and incubating them for 15 minutes. After the incubation, wash the culture twice with 1 milliliter of PBS. Add 500 microliters of PBS with 3% BSA and 0.1% Triton X-100 to permeabilize the culture. Incubate the tube for five minutes, then wash the organoids three times with 1 milliliter of PBS and 3% BSA.

Next, incubate the culture with primary antibodies for one hour, then repeat the washes with PBS and BSA. Incubate with the secondary antibodies and repeat the washes, making sure to aspirate and discard all supernatant.

To image the spheroids, add 5 microliters of mounting medium containing DAPI, and use a P20 pipette to transfer 5 microliters of the spheroids to a glass slide. Seal the slide with a cover slip, and image with a fluorescence microscope using the 10, 20, and 40 times objectives.