Abstract
Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples.
Highlights
IntroductionBiological tissues and organs present a complex three-di mensional structure
Human cerebral organoids were generated according to a protocol from Lancaster et al (2013) with small modifications. 2- and 3-month-old cerebral organoids were used for tissue clearing protocol
Cerebral organoids are a unique novel technology that allows the reconstruction of early human neurogenesis
Summary
Biological tissues and organs present a complex three-di mensional structure. Due to their opacity and high level of autofluorescence, three-dimensional reconstruction of such objects is an extremely laborious, but necessary process. On the other hand, some of the protocols involve using toxic and corrosive chemicals that require special objectives to avoid damage to the microscope or require other special equipment. Most of these protocols have been developed to clarify entire organs or their big fragments
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