Abstract
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.
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Acknowledgments
My work in this area has been supported primarily by grants from the National Health and Medical Research Council of Australia to me and to Judy Morris. The development of multiple-labelling immunofluorescence in whole mounts was initiated by Marcello Costa and John Furness. Since then, many laboratory members and colleagues, especially Judy Morris and Sue Matthew, have added to the development and application of the methods described here, but Pat Vilimas has been a critical contributor throughout.
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Gibbins, I.L. (2012). Multiple Immunohistochemical Labelling of Peripheral Neurons. In: Badoer, E. (eds) Visualization Techniques. Neuromethods, vol 70. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-897-9_1
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DOI: https://doi.org/10.1007/978-1-61779-897-9_1
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Publisher Name: Humana Press, Totowa, NJ
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