Stereological techniques that estimate cell numbers require specific training and elaborate sampling strategies to infer total numbers of cells in well-defined structures of measurable volume. The isotropic fractionator is a fast and inexpensive method that requires little specific training and few materials before it can be used to quantify total numbers of neuronal and nonneuronal cells in the whole brain or any dissectible regions thereof. It consists in transforming highly anisotropic (paraformaldehyde fixed and dissected) brain structures into homogeneous, isotropic suspensions of cell nuclei which can be counted and identified morphologically and immunocytochemically as neuronal or nonneuronal. Estimates of total cell, neuronal and nonneuronal, numbers can be obtained within 24 h, and vary by less than 10% among samples of the same structure. Since the estimates obtained are independent of brain volume, they can be used in comparative studies of brain volume variation among species and in studies of phylogenesis, development, adult neurogenesis, and pathology.
Numbers of neurons Brain size Isotropic fractionator Allometry NeuN Stereology
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Thanks to Roberto Lent for encouraging and supporting the creation of this method, to Jon Kaas for continued support, to Christine Collins for improving the method with the FACS variation, and to Paul Manger for insights on tissue storage. The development and improvement of this method were made possible by grants from CNPq and FAPERJ.
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