Three-dimensional reconstruction of transmitter-identified central neurons by “en bloc” immunofluorescence histochemistry and confocal scanning microscopy

A new method for three-dimensional reconstruction of transmitter-identified neurons is presented which involves “en bloc” immunofluorescence histochemistry and confocal scanning microscopy. The technique was applied to different types of neurons in the rat brain and lamprey spinal cord. Thick sections or tissue “blocs” (50–200 μm thick) were incubated with antisera against neuropeptides or monoaminergic markers, followed by fluorescent secondary antibodies. Three-dimensional reconstructions were obtained by scanning the preparations in sequential focal planes with a thin laser beam, while sampling the emitted light in each focal plane. The method is convenient and can be applied to a wide variety of neuron types. The reconstructions obtained are accurate since the “optical serial sections” of the specimen are perfectly aligned, and optic disturbances such as “halo” phenomena do not occur.