A Specialized Method to Resolve Fine 3D Features of Astrocytes in Nonhuman Primate (Marmoset, Callithrix jacchus) and Human Fixed Brain Samples
Astrocytes are among the most numerous cells in the brain and fulfill diverse functions in homeostasis and regulation of neuronal activity. Astrocytes also dramatically change their properties in response to brain injury or disease, a process called reactive gliosis. Precisely how astrocytes contribute to healthy brain function and play differential roles in brain pathology and regeneration remain important areas of investigation. To better understand the properties of astrocytes, more sophisticated approaches for probing their rich and complex anatomical and molecular features are needed to fully determine their contribution to brain physiology. Here we present an efficient and straightforward immunolabeling protocol to obtain high-resolution fluorescence-based images from fixed nonhuman primate (common marmoset Callithrix jacchus) and human brain samples. Importantly, the protocol is useful for obtaining images from samples that have been stored in fixative solutions (such as formalin) for years. This approach is especially useful for three-dimensional, multichannel confocal microscopy and can be optimized for super-resolution techniques such as stimulated emission depletion (STED) microscopy. We also present a strategy for using specific combinations of markers to define the phenotypic variations and cellular/subcellular properties of astrocytes to better predict the function of these cells on their surrounding brain microenvironment.
Key wordsImmunofluorescence Confocal microscopy STED Antibodies Astrocytes Human brain Nonhuman primate 3D Disease
This work was supported by the Espoir-en-tête Rotary International award and the Auguste et Simone Prévot foundation (to D.S.B.). This work was supported by the Postdoctoral Training (Applicants living outside of Québec) of the Fonds de Recherche du Québec-Santé (FRQS) Canada (to G.Q.). This work was supported by the Canadian Institutes of Health Research (MOP 111152, PJT148569, PJT156247 to K.K.M.); Natural Sciences and Engineering Research Council of Canada (408044-2011 and 69404 to K.K.M.); Canada Research Chairs Program (K.K.M.); Brain Canada/W. Garfield Weston Foundation (K.K.M.). We thank the Douglas-Bell Canada Brain Bank (Douglas Mental Health University Institute, Montréal, QC, Canada) for providing the human samples and the Molecular Imaging Platform at the Research Institute of the McGill University of Health Centre for instrumentation and support.
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