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A Specialized Method to Resolve Fine 3D Features of Astrocytes in Nonhuman Primate (Marmoset, Callithrix jacchus) and Human Fixed Brain Samples

Part of the Methods in Molecular Biology book series (MIMB,volume 1938)


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 words

  • Immunofluorescence
  • Confocal microscopy
  • STED
  • Antibodies
  • Astrocytes
  • Human brain
  • Nonhuman primate
  • 3D
  • Disease

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  • DOI: 10.1007/978-1-4939-9068-9_6
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  1. Vasile F, Dossi E, Rouach N (2017) Human astrocytes: structure and functions in the healthy brain. Brain Struct Funct 222(5):2017–2029.

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  2. Farmer WT, Murai K (2017) Resolving astrocyte heterogeneity in the CNS. Front Cell Neurosci 11:300

    CrossRef  Google Scholar 

  3. Oberheim NA, Wang X, Goldman S, Nedergaard M (2006) Astrocytic complexity distinguishes the human brain. Trends Neurosci 29(10):547–553

    CAS  CrossRef  Google Scholar 

  4. Sofroniew MV, Vinters HV (2010) Astrocytes: biology and pathology. Acta Neuropathol 119(1):7–35

    CrossRef  Google Scholar 

  5. Bouvier DS, Jones EV, Quesseveur G, Davoli MA, AF T, Quirion R, Mechawar N, Murai KK (2016) High resolution dissection of reactive glial nets in alzheimer's disease. Sci Rep 6:24544

    CAS  CrossRef  Google Scholar 

  6. Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K (2013) Structural and molecular interrogation of intact biological systems. Nature 497(7449):332–337

    CAS  CrossRef  Google Scholar 

  7. Lai HM, Liu AKL, Ng HHM, Goldfinger MH, Chau TW, DeFelice J, Tilley BS, Wong WM, Wu W, Gentleman SM (2018) Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues. Nat Commun 9(1):1066

    CrossRef  Google Scholar 

  8. Yang Z, Wang KK (2015) Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker. Trends Neurosci 38(6):364–374

    CAS  CrossRef  Google Scholar 

  9. Schubert V, Bouvier D, Volterra A (2011) SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis. Glia 59(10):1472–1488

    CrossRef  Google Scholar 

  10. Hubbard JA, Szu JI, Yonan JM, Binder DK (2016) Regulation of astrocyte glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4) expression in a model of epilepsy. Exp Neurol 283(Pt A):85–96

    CAS  CrossRef  Google Scholar 

  11. Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28(1):264–278

    CAS  CrossRef  Google Scholar 

  12. Bittar PG, Charnay Y, Pellerin L, Bouras C, Magistretti PJ (1996) Selective distribution of lactate dehydrogenase isoenzymes in neurons and astrocytes of human brain. J Cereb Blood Flow Metab 16(6):1079–1089

    CAS  CrossRef  Google Scholar 

  13. Orr AG, Hsiao EC, Wang MM, Ho K, Kim DH, Wang X, Guo W, Kang J, Yu GQ, Adame A, Devidze N, Dubal DB, Masliah E, Conklin BR, Mucke L (2015) Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory. Nat Neurosci 18(3):423–434

    CAS  CrossRef  Google Scholar 

  14. Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW (2011) Sharper low-power STED nanoscopy by time gating. Nat Methods 8(7):571–573

    CAS  CrossRef  Google Scholar 

  15. Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19(11):780–782

    CAS  CrossRef  Google Scholar 

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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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Correspondence to Keith K. Murai or David S. Bouvier .

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Quesseveur, G., Fouquier d’Hérouël, A., Murai, K.K., Bouvier, D.S. (2019). A Specialized Method to Resolve Fine 3D Features of Astrocytes in Nonhuman Primate (Marmoset, Callithrix jacchus) and Human Fixed Brain Samples. In: Di Benedetto, B. (eds) Astrocytes. Methods in Molecular Biology, vol 1938. Humana Press, New York, NY.

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  • Print ISBN: 978-1-4939-9067-2

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