Light Microscopy pp 137-148

Part of the Methods in Molecular Biology book series (MIMB, volume 689)

Using the Fluorescent Styryl Dye FM1-43 to Visualize Synaptic Vesicles Exocytosis and Endocytosis in Motor Nerve Terminals

  • Ernani Amaral
  • Silvia Guatimosim
  • Cristina Guatimosim
Protocol

Abstract

The styryl dye FM1-43 is a powerful tool to track exocytosis, endocytosis and recycling of secretory granules or vesicles. Due to its unique structure, dye molecules reversibly partition into the outer leaflet of surface membrane without permeating due to two cationic charges located in their headgroup. When a secretory cell is stimulated to evoke exocytosis, FM1-43 molecules that were inserted in the membrane are internalized during compensatory endocytosis and newly formed secretory granules or vesicles become stained with dye (staining/endocytosis). If stained secretory granules or vesicles undergo exocytosis in dye-free medium, due to concentration gradient, FM1-43 molecules dissociate from the membrane and loose fluorescence (destaining/exocytosis). Using a fluorescence microscope attached to a CCD camera or a confocal, it is possible to follow secretion in live cell or tissue preparations and in this chapter, we will make a description of FM1-43 staining and destaining protocol using the neuromuscular junction as experimental model. This technique has allowed answering important questions concerning synaptic vesicle recycling, which is a key step for neuronal communication. In addition, FM1-43 has proven to be an excellent tool for investigating membrane internalization and endosome recycling in a variety of cell types.

Key words

FM1-43 neuromuscular junction fluorescence microscopy exocytosis endocytosis synaptic vesicles 

Copyright information

© Humana Press 2011

Authors and Affiliations

  • Ernani Amaral
    • 1
  • Silvia Guatimosim
    • 2
  • Cristina Guatimosim
    • 1
  1. 1.Department of Morphology, ICBFederal University of Minas GeraisBelo HorizonteBrazil
  2. 2.Department of Physiology and Biophysics, ICBFederal University of Minas GeraisBelo HorizonteBrazil

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