Background: NeuN and Nissl staining (toluidine blue, cresyl violet staining) are routinely used methods in unbiased stereological estimation of the total number of hippocampal neurons.
New method: In the present study, we stained serial frozen coronal sections from 5 normal adult male Sprague-Dawley rat brains with different methods, measured the deformation of hippocampal area in brain sections and estimated the total number of hippocampal neurons using the optical fractionator.
Results: The deformation in x, y-axis was not obviously different with different staining protocols, but shrinkage in z-axis was significant after staining (p < 0.001). NeuN staining produced significant higher estimate number than cresyl violet staining by 24% (p = 0.002), however, NeuN and Cresyl Violet staining showed a high degree of correlation in quantification of total neuronal numbers and both methods are suitable for unbiased stereological estimation.
Comparison with existing method (s): NeuN is more reliable but if time is limited or the number of animals used in experiments is high, cresyl violet staining may be a feasible method.
Conclusions: Compared with previous estimates of the neurons number in rat hippocampus, our present data is reliable and the stereological analysis based on our system is a cost-effective unbiased method for estimation of neuron number.
Keywords: Hippocampal neuron; Histological study; Stereological analysis; Unbiased estimation.
Copyright © 2015 Elsevier B.V. All rights reserved.