| First Author | Aryal SP | Year | 2021 |
| Journal | Curr Protoc | Volume | 1 |
| Issue | 10 | Pages | e279 |
| PubMed ID | 34694747 | Mgi Jnum | J:361077 |
| Mgi Id | MGI:7664699 | Doi | 10.1002/cpz1.279 |
| Citation | Aryal SP, et al. (2021) Characterization of Astrocyte Morphology and Function Using a Fast and Reliable Tissue Clearing Technique. Curr Protoc 1(10):e279 |
| abstractText | Astrocytic processes interact with synapses throughout the brain modulating neurotransmitter signaling and synaptic communication. During conditions such as exposure to drugs of abuse and neurological diseases, astrocytes respond by altering their morphological and functional properties. Reactive astrocyte phenotypes exhibit a bushy morphology with altered soma volume and an increased number of processes compared to resting astrocytes. The reactive astrocytic phenotype also overexpresses proteins one of which can be glial fibrillary acidic protein (GFAP). Fluorescence microscopy on thin tissue sections (<20 microm) requires reconstruction, often through multiple sections, to delineate the full astrocytic morphology. In contrast, tissue clearing methods have been developed that enable imaging of larger sections including the whole brain, providing an opportunity to see in-depth changes in single cell structure. In this article, a detailed protocol for studying astrocyte morphology using tissue clearing and subsequent imaging of whole brains as well as region-specific slices is provided. This method is ideal for understanding the effect of different physiological conditions on astrocyte morphology. A standard biochemistry laboratory has the resources to accomplish tissue clearing using this protocol and most universities have the required imaging facilities. Protocols to study brains from both genetically modified mice that contain an astrocyte-specific marker and from wild-type mice using antibody labeling steps after tissue clearing are provided. We also describe general protocols to conduct fluorescence imaging of astrocytes in cleared tissue to characterize their morphology. This protocol could be useful for researchers working in the rapidly growing field of astrocyte biology. (c) 2021 Wiley Periodicals LLC. Basic Protocol 1: Brain perfusion, fixation, and tissue clearing Alternate Protocol: Clearing brain tissue with passive clarity Basic Protocol 2: Antibody labeling and refractive index matching Basic Protocol 3: Fluorescence imaging and characterization of astrocyte morphology. |
