We therefore compared cell counts obtained using immunophenotyping with a modified Giemsa staining method that stains all cells. Many IHC markers are specific but not always sensitive. First, we wish to present a comprehensive tool to validate IHC marker for stereological application.
Commonly used markers, although not all have been used in stereology, include NeuN for neocortical neurons glial fibrillary acidic protein (GFAP) – and glutamine synthetase (GS) for astrocytes CNPase, a member of the cyclic nucleotide phosphodiesterase family membrane-bound enzyme expressed by oligodendrocytes and CD11b, an integrin found in microglia. The combination of IHC and stereology in quantitative analysis of the brain utilizes cell-type-specific antibodies that target diverse neurochemical properties of neuronal and glial cell populations. Although these validation methods are useful first steps to ensure specificity, they do not guarantee sufficient sensitivity to ensure all cells of interest are included in the final count in histological preparations suitable for stereological estimation of cell number. This is of particular interest in research projects where exact numbers are important for further decision making, as opposed to routine pathological diagnosis or qualitative studies where the total cell number is not the specific goal.Ī variety of approaches can be used to demonstrate that IHC antibodies are specific, including western blot analysis combined with mass spectrometry or crystallography, staining of cells from known in vitro cell lines, and qualitative assessment of tissue samples –.
Therefore, staining specific cell types will prove valuable in some circumstances.ĭespite widespread use of immunohistochemistry (IHC) in experimental neuroscience and neuropathology, only a few studies published in peer-reviewed journals have validated that the antibody used actually stains the total population of the specific cell type of interest. Such methods of cell categorization may be subject to intra- and interobserver variability. Numerous studies using design-based stereology have attempted to quantitatively evaluate these changes, and most of these have identified cells using morphological criteria –. Histology is a preferred method for evaluating morphological changes in neuropathology.
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