High-resolution 3D imaging of fixed and cleared organoids
- Author(s)
- Dekkers, JF; Alieva, M; Wellens, LM; Ariese, HCR; Jamieson, PR; Vonk, AM; Amatngalim, GD; Hu, H; Oost, KC; Snippert, HJG; Beekman, JM; Wehrens, EJ; Visvader, JE; Clevers, H; Rios, AC;
- Journal Title
- Nature Protocols
- Publication Type
- Journal Article in press
- Abstract
- In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.
- Publisher
- Springer Nature
- Research Division(s)
- Cancer Biology And Stem Cells
- PubMed ID
- 31053799
- Publisher's Version
- https://doi.org/10.1038/s41596-019-0160-8
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2019-05-08 09:28:15
Last Modified: 2019-05-08 09:32:30