An empirical analysis of the precision of estimating the numbers of neurons and glia in human neocortex using a fractionator-design with sub-sampling

Improving histomorphometric analysis of the human neocortex by combining stereological cell counting with immunohistochemical visualisation of specific neuronal and glial cell populations is a methodological challenge. To enable standardized immunohistochemical staining, the amount of brain tissue to be stained and analysed by cell counting was efficiently reduced using a fractionator protocol involving several steps of sub-sampling. Since no mathematical or statistical tools exist to predict the variance originating from repeated sampling in complex structures like the human neocortex, the variance at each level of sampling was determined empirically. The methodology was tested in three brains analysing the contribution of the multi-step sampling procedure to the precision on the estimated total numbers of immunohistochemically defined NeuN expressing (NeuN(+)) neurons and CD45(+) microglia. The results showed that it was possible, but not straight forward, to combine immunohistochemistry and the optical fractionator for estimation of specific subpopulations of brain cells in human neocortex.