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Animal models are widely used to improve our understanding of the complex pathophysiological processes underlying diseases of the central nervous system (CNS), as well as a providing a means of evaluating the efficacy of new therapeutic agents. The advent of high-resolution, dedicated small animal magnetic resonance imaging and positron emission tomography scanners has greatly improved the value of animal imaging for such studies. However, the use of in vivo imaging markers requires extensive validation against gold standard, ex vivo tissue studies. In this paper, we describe methods for three-dimensional reconstruction of two-dimensional serial histological sections to create volumetric data, a major step in the use of ex vivo data for validating in vivo imaging techniques.