Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Aug 2;126(4):901–910. doi: 10.1083/jcb.126.4.901

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

PMCID: PMC2120127  PMID: 7519623

Abstract

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

Full Text

The Full Text of this article is available as a PDF (4.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balercia G., Chen S., Bentivoglio M. Electron microscopic analysis of fluorescent neuronal labeling after photoconversion. J Neurosci Methods. 1992 Oct-Nov;45(1-2):87–98. doi: 10.1016/0165-0270(92)90046-g. [DOI] [PubMed] [Google Scholar]
  2. Bentivoglio M., Su H. S. Photoconversion of fluorescent retrograde tracers. Neurosci Lett. 1990 May 31;113(2):127–133. doi: 10.1016/0304-3940(90)90291-g. [DOI] [PubMed] [Google Scholar]
  3. Carter K. C., Bowman D., Carrington W., Fogarty K., McNeil J. A., Fay F. S., Lawrence J. B. A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. Science. 1993 Feb 26;259(5099):1330–1335. doi: 10.1126/science.8446902. [DOI] [PubMed] [Google Scholar]
  4. Carter K. C., Taneja K. L., Lawrence J. B. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. doi: 10.1083/jcb.115.5.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Conrad P. A., Nederlof M. A., Herman I. M., Taylor D. L. Correlated distribution of actin, myosin, and microtubules at the leading edge of migrating Swiss 3T3 fibroblasts. Cell Motil Cytoskeleton. 1989;14(4):527–543. doi: 10.1002/cm.970140410. [DOI] [PubMed] [Google Scholar]
  6. Hsu S. M., Raine L., Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981 Apr;29(4):577–580. doi: 10.1177/29.4.6166661. [DOI] [PubMed] [Google Scholar]
  7. Hulspas R., Krijtenburg P. J., Keij J. F., Bauman J. G. Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy. J Histochem Cytochem. 1993 Aug;41(8):1267–1272. doi: 10.1177/41.8.7687265. [DOI] [PubMed] [Google Scholar]
  8. Lübke J. Photoconversion of diaminobenzidine with different fluorescent neuronal markers into a light and electron microscopic dense reaction product. Microsc Res Tech. 1993 Jan 1;24(1):2–14. doi: 10.1002/jemt.1070240103. [DOI] [PubMed] [Google Scholar]
  9. Maranto A. R. Neuronal mapping: a photooxidation reaction makes Lucifer yellow useful for electron microscopy. Science. 1982 Sep 3;217(4563):953–955. doi: 10.1126/science.7112109. [DOI] [PubMed] [Google Scholar]
  10. Pagano R. E., Sepanski M. A., Martin O. C. Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy. J Cell Biol. 1989 Nov;109(5):2067–2079. doi: 10.1083/jcb.109.5.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Papadopoulos G. C., Dori I. DiI labeling combined with conventional immunocytochemical techniques for correlated light and electron microscopic studies. J Neurosci Methods. 1993 Mar;46(3):251–258. doi: 10.1016/0165-0270(93)90074-2. [DOI] [PubMed] [Google Scholar]
  12. Peters D. M., Portz L. M., Fullenwider J., Mosher D. F. Co-assembly of plasma and cellular fibronectins into fibrils in human fibroblast cultures. J Cell Biol. 1990 Jul;111(1):249–256. doi: 10.1083/jcb.111.1.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sandell J. H., Masland R. H. Photoconversion of some fluorescent markers to a diaminobenzidine product. J Histochem Cytochem. 1988 May;36(5):555–559. doi: 10.1177/36.5.3356898. [DOI] [PubMed] [Google Scholar]
  14. Schmued L. C., Snavely L. F. Photoconversion and electron microscopic localization of the fluorescent axon tracer fluoro-ruby (rhodamine-dextran-amine). J Histochem Cytochem. 1993 May;41(5):777–782. doi: 10.1177/41.5.7682231. [DOI] [PubMed] [Google Scholar]
  15. Takizawa P. A., Yucel J. K., Veit B., Faulkner D. J., Deerinck T., Soto G., Ellisman M., Malhotra V. Complete vesiculation of Golgi membranes and inhibition of protein transport by a novel sea sponge metabolite, ilimaquinone. Cell. 1993 Jun 18;73(6):1079–1090. doi: 10.1016/0092-8674(93)90638-7. [DOI] [PubMed] [Google Scholar]
  16. von Bartheld C. S., Cunningham D. E., Rubel E. W. Neuronal tracing with DiI: decalcification, cryosectioning, and photoconversion for light and electron microscopic analysis. J Histochem Cytochem. 1990 May;38(5):725–733. doi: 10.1177/38.5.2185313. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES