A method for combining confocal and electron microscopic examination of sections processed for double- or triple-labelling immunocytochemistry.

Double-labelling immunocytochemical techniques are important for revealing synaptic connections between different populations of neurons within the central nervous system. This article describes a new method in which confocal laser scanning microscopy and electron microscopy are performed on the same Vibratome section which has been processed for immunocytochemistry. Two or three primary antibodies are initially detected with fluorescent secondary antibodies and observed with the confocal microscope. The primary antibodies are then revealed by an immunoperoxidase technique (with diaminobenzidine), and the material is prepared for electron microscopy. By comparing the resulting electron micrographs with the images acquired from the confocal microscope, it is possible to recognise each immunoreactive structure seen with the electron microscope in the original confocal images, and therefore to determine which type(s) of immunoreactivity each structure contains. This method has been used to demonstrate that some neurons in the spinal dorsal horn which possess the neurokinin-1 receptor receive axosomatic synapses from boutons that contain substance P and calcitonin gene-related peptide.