Immunofluorescent Staining of Spheroids: A Technique to Analyze Spheroids for Expression of Intra- and Extracellular Antigen Expression

- Spheroids are three-dimensional in vitro cell culture models. For immunofluorescent staining, first, generate a spheroid culture of the required cell type in a multiwell plate. Using a micropipette with a wide orifice tip, collect the spheroids, taking care to avoid rupturing due to shear forces.

Next, fix and permeabilize the spheroids to render individual cells permeable. Now, incubate with a solution containing proteins to block non-specific protein interaction sites on the cell surface. Treat the spheroids with a desired primary antibody cocktail. Incubate for the antibodies to bind to the target molecules on the cell surface.

Next, label the spheroids with a fluorescently labeled secondary antibody solution specific to the primary antibodies. Finally, treat the spheroids with a suitable fluorescent DNA-binding dye to stain cell nuclei. Using a laser-scanning fluorescence microscope, image multiple optical cross-sections of the spheroid at different optical planes.

The captured images reflect fluorescently-labeled nuclei and antigens of interest. Merge the stacks using relevant software to obtain the final composite image of the 3D spheroid. In the following protocol, we will