Use of cryostat sections from snap-frozen nervous tissue for combining stereological estimates with histological, cellular, or molecular analyses on adjacent sections

Adequate tissue preparation is essential for both modern stereological and immunohistochemical investigations. However, combining these methodologies in a single study presents a number of obstacles pertaining to optimal hist ological preparation. Tissue shrinkage and loss of nuclei/nucleoli from the unprotected section surfaces of unembedded tissue used for immunohistochem istry may be problematic with regard to adequate stereological design. In t his study, frozen cryostat sections from hippocampal and cerebellar regions of two rat strains and cerebellar and cerebral regions from a human brain were analyzed to determine the potential impact of these factors on estimat es of neuron number obtained using the optical disector. Neuronal nuclei an d nucleoli were clearly present in thin sections of snap-frozen rat (3 mum) and human (6 mum) tissue, indicating that neuronal nuclei/nucleoli are not unavoidably lost from unprotected section surfaces of unembedded tissue. I n order to quantify the potential impact of any nuclear loss, optical fract ionator estimates of rat hippocampal pyramidal cells in areas CA1-3 and cer ebellar granule and Purkinje cells were made using minimal (1 mum) upper gu ard zones. Estimates did nut differ from data reported previously in the li terature. This data indicates that cryostat sections of snap-frozen nervous tissue may successfully be used for estimating total neuronal numbers usin g optical disectors. (C) 2000 Elsevier Science B.V. All rights reserved.