Cell Staining: Fluorescent Labelling of the Golgi Apparatus

Selma Y Dejgaard, McGill University, Montreal, Quebec, Canada
Kurt Dejgaard, McGill University, Montreal, Quebec, Canada
John F Presley, McGill University, Montreal, Quebec, Canada

Published online: December 2010

DOI: 10.1002/9780470015902.a0002633.pub2

Immunofluorescence has been the primary method for labelling the Golgi apparatus for light microscopic observation in fixed cells. The Golgi apparatus was initially labelled in living cells by using that the fluorescent ceramide analogue N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine or by microinjection of fluorescently conjugated antibodies to exposed Golgi epitopes. With the common availability of the green fluorescent protein (GFP) and advanced fluorescence microscopes including confocal microscopes, imaging of the Golgi apparatus in living cells is now a major experimental approach. Time-lapse imaging of living cells has also been combined with photobleach techniques in order to study the dynamics of transient protein association with the Golgi apparatus. Similar approaches have been used to study the dynamics of the cargo transport through the exocytic pathway.

Key Concepts:

  • Immunofluorescence can be used to visualise proteins in the Golgi apparatus or its subdomains.
  • Live-cell visualisation of the Golgi apparatus is possible with GFP-tagged proteins.
  • Photobleaching and photoactivation techniques permit visualisation of protein dynamics and flux through the Golgi apparatus in living cells.

Keywords: Golgi apparatus; exocytosis; immunofluorescence; vital staining; GFP; photobleach; fluorescence microscopy

Figure 1. (a) Normal rat kidney (NRK) cells stained with (left) rabbit antibody directed against the cis-Golgi marker p115 and a Cy5-tagged secondary antibody transfected with (middle) GFP-tagged Rab33b, a cytosolic GTPase localising to the medial-Golgi; and (right) the TGN marker TGN38 visualised with a mouse monoclonal antibody and a Cy3-tagged secondary antibody localising to the trans-cisternae of the Golgi and the trans-Golgi network. (b) Merged images show that the localisation of the proteins does not overlap completely. p115 (blue), Rab33b (green) and TGN38 (red).
Figure 2. COS7 cells transfected with GFP-p58/ERGIC53 which localises to the endoplasmic reticulum, Golgi apparatus and intermediate compartment. (a) Photobleach sequence described in the text. The region containing the Golgi apparatus and Golgi-associated intermediate compartment was bleached and recovery visualised as indicated. (b) Quantitation of fluorescence recovery in the Golgi region in a photobleach experiment. Fluorescence in the Golgi region is normalised to total cell fluorescence. The initial point immediately preceeds the bleach. Note that in this experiment a plateau is reached below the initial prebleach level, indicating some immobile fraction.
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 References
    Dejgaard YS, Murshid A, Dee KM and Presley JF (2007) Confocal microscopy-based linescan methodologies for intra-Golgi localization of proteins. Journal of Histochemistry & Cytochemistry 55(7): 709–719.
    Golgi C (1898) Sur la structure des cellules nerveuses. Archives Italiennes de Biologie 30: 60–71.
    Hirschberg K, Miller CM, Ellenberg J et al. (1998) Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport intermediates in living cells. Journal of Cell Biology 143: 1485–1503.
    Pagano RE, Martin OC, Kang HC and Haugland RP (1991) A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. Journal of Cell Biology 110: 1267–1279.
    book Presley JF (2005) "Measurement of protein motion by photobleaching". In: Stephens D (ed.) Cell Imaging: Methods Express, pp. 119–144. Oxford: Scion Publishing.
    Stephens DJ and Allan VJ (2003) Light microscopy techniques for live cell imaging. Science 300: 82–86.
 Further Reading
    book Inoue S and Spring KS (1997) Video Microscopy: The Fundamentals, 2nd edn. New York: Plenum Press.
    book Pawley JB (1995) Handbook of Biological Confocal Microscopy, 2nd edn. New York: Plenum Press.

Related Sub-Topics:

Cell Compartments and Cellular Organelles | Techniques in Cell Biology | Optical Microscopy
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Article Title: Cell Staining: Fluorescent Labelling of the Golgi Apparatus
 
How to Cite close
Dejgaard, Selma Y, Dejgaard, Kurt, and Presley, John F(Dec 2010) Cell Staining: Fluorescent Labelling of the Golgi Apparatus. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002633.pub2]