Multiple Immunohistochemical Labelling of Peripheral Neurons

This chapter describes well-established procedures for multiple-labelling immunofluorescence as applied to peripheral neurons. Tissues are fixed with a mixture of formaldehyde and picric acid, and then processed through solvents (ethanol and dimethyl sulfoxide or xylene) before preparation as sections (frozen or embedded in polyethylene glycol) or whole mounts. Specimens are exposed to small volumes of antibody mixtures and are then mounted in buffered glycerol prior to examination with fluorescent microscope fitted with appropriate optics for multi-labelling fluorescence. Critical controls are summarised for all stages of the process, including the specificity of the primary antibodies, the secondary antibodies, and the subsequent image acquisition and analysis.