Whole-mount Immunofluorescence Imaging: A Fixing and Staining Technique to Evaluate Intact Organoids
Trascrizione
Prostate organoids primarily constitute an outer layer of basal epithelial cells and inner layers of luminal epithelial cells arranged around a central lumen.
Whole-mount imaging allows the study of intact 3D organoids while maintaining their morphology and heterogeneity. To begin staining, treat the organoids with a suitable fixative to lock their cellular components in place, thereby preserving them. Wash the sample to remove excess fixatives.
Incubate with a cocktail of blocking solution and DNA-staining dye. Proteins in the blocking solution passively bind to non-specific sites of the cells and reduce any background staining. Concurrently, the DNA-staining dye stains the cells' nuclei.
Add two sets of primary antibodies targeting specific antigens expressed by the two constituent cell populations. Incubate with two different fluorescent-dye-conjugated secondary antibodies that bind to the primary antibodies. Wash the sample to remove any unbound antibodies.
Immerse the stained organoids sequentially in increasing concentrations of surfactant-containing sugar solutions to improve tissue transparency without compromising the organoid architecture. This treatment enhances the imaging depth of the sample.
Mount the organoids on a microscope slide carrying spacers to preserve their 3D morphology while imaging. Use a confocal microscope to observe the fluorescence emissions from the basal and luminal cell populations arranged around the central lumen.
