Immunocytochemical Analysis of Neuronal Differentiation

Fully understanding the phenotype of neurons in vivo involves examining their morphology, immunocytochemically analyzing their protein expression, examining their afferent and efferent integration into neuronal microcircuitry, and functionally examining their activity. This task is significantly more difficult when you are attempting to determine whether multipotent precursor cells, often referred to as stem cells, differentiate into neurons in vivo. Transplanted or endogenous precursor cells often produce relatively small numbers of new neurons in the adult brain, making electron microscopy or electrophysiological analysis extremely challenging, and functional analysis difficult. Studying such cells usually depends heavily on immunocytochemical approaches. We review a range of immunocytochemical techniques for identifying whether transplanted or endogenous neural precursors have differentiated into mature neurons. We provide immunocytochemical protocols for the migratory neuronal marker Doublecortin (Dcx), the early expressed marker Hu, and mature neuronal marker NeuN. In Chapters 25 and 27 of Part IV, we provide protocols for identifying newborn cells by using the mitotic label bromodeoxyuridine and for examining axonal projections by using the retrograde label FluoroGold.