Dual fluorescence combined with a two-color immunoperoxidase technique: A new way of visualizing diverse neuronal elements

A method is described that allows an estimation of the neurotransmitter-related immunoreactivity, morphology and relationship to other immunoreactive elements, of single functionally identified neurons in the central nervous system. First, neurons are identified electrophysiologically using intracellular recording and labelled by iontophoresis of lucifer yellow (LY). After fixation and sectioning of the brain tissue, the location of the labelled neuron is determined by fluorescence microscopy. Sections are then processed using an indirect immunofluorescence procedure in order to determine the antigen content of the labelled neurons. Antisera to LY and an avidin-biotin immunoperoxidase technique is then used to localize LY in a permanent form, while the other previously localized antigen is permanently visualised by using the fluorescent-labelled second antibody as a bridge antibody in a peroxidase anti-peroxidase technique. The method is illustrated by an examination of neurons in the medulla oblongata of the rat, that have been stained intracellularly with LY, their content of tyrosine hydroxylase assessed, and their relationship to other tyrosine hydroxylase immunoreactive neurons determined.