Photoconversion of CFP to Study Neuronal Tissue with Electron Microscopy

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1148)

Abstract

Being able to use versatile light microscopy on live or fixed samples followed by electron microscopy imaging for high resolution analyses is a challenging goal. The advantage is of course that tracing and localizing fluorescently labeled molecules yields great information about dynamic cellular processes, while electron microscopy of the same sample provides exquisite information about subcellular structures. Here, I describe the straightforward combination of both methods by photoconversion of diaminobenzidine (DAB) through cyan fluorescent protein (CFP) tagged proteins localized to the Golgi apparatus in primary hippocampal neurons.

Key words

CFP DAB Photoconversion Neuron Transmission electron microscopy 
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19(11):555–565PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Ward TH, Lippincott-Schwartz J (2006) The uses of green fluorescent protein in mammalian cells. Methods Biochem Anal 47:305–337PubMedGoogle Scholar
  3. 3.
    Giepmans BN, Adams S R, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312(5771):217–224PubMedCrossRefGoogle Scholar
  4. 4.
    Grabenbauer M et al (2005) Correlative microscopy and electron tomography of GFP through photooxidation. Nat Methods 2(11): 857–862PubMedCrossRefGoogle Scholar
  5. 5.
    Pellicciari C et al (2013) Ultrastructural detection of photosensitizing molecules by fluorescence photoconversion of diaminobenzidine. Histochem Cell Biol 139(6):863–871PubMedCrossRefGoogle Scholar
  6. 6.
    Harata N, Ryan TA, Smith SJ, Buchanan J, Tsien RW (2001) Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion. Proc Natl Acad Sci U S A 98(22):12748–12753PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Opazo F, Rizzoli SO (2010) Studying synaptic vesicle pools using photoconversion of styryl dyes. J Vis Exp. 36Google Scholar
  8. 8.
    Dresbach T et al (2003) Functional regions of the presynaptic cytomatrix protein bassoon: significance for synaptic targeting and cytomatrix anchoring. Mol Cell Neurosci 23(2):279–291PubMedCrossRefGoogle Scholar
  9. 9.
    Ohki EC, Tilkins ML, Ciccarone VC, Price PJ (2001) Improving the transfection efficiency of post-mitotic neurons. J Neurosci Methods 112(2):95–99PubMedCrossRefGoogle Scholar
  10. 10.
    Fahimi HD, Baumgart E (1999) Current cytochemical techniques for the investigation of peroxisomes. A review. J Histochem Cytochem 47(10):1219–1232PubMedCrossRefGoogle Scholar
  11. 11.
    Meiblitzer-Ruppitsch C et al (2008) Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion. Histochem Cell Biol 130(2):407–419PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Center of Anatomy, Department Anatomy and EmbryologyUniversity Medical Center GöttingenGöttingenGermany

Personalised recommendations