Multifluorescence Labeling Techniques and Confocal Laser Scanning Microscopy on Lung Tissue

  • Maria Stern
  • Douglas J. Taatjes
  • Brooke T. Mossman
Part of the Methods in Molecular Biology™ book series (MIMB, volume 319)


Lung tissue consists of more than 40 individual cell types that might interact to produce adverse pathologies. After injury, a number of signaling proteins expressed in various epithelial and other cell types have been linked to the advent of apoptosis, compensatory proliferation, and adaptation to stress. We describe here the use of immunochemistry and multifluorescence approaches using confocal laser scanning microscopy to define the signaling pathways (protein kinases C and mitogen-activated protein kinases) activated by asbestos fibers after inhalation. Using these approaches, we are able to localize signaling events in distinct cell types of the lung and determine their status in the cell cycle (resting or nonresting). Moreover, we are able to determine whether various signaling proteins colocalize in cells and the sites affected by asbestos fibers.

Key Words

Immunofluorescence confocal laser scanning microscopy asbestos extracellular signal-regulated kinases (ERKs) PKCδ proliferation marker Ki-67. 


  1. 1.
    Taatjes, D. J., Palmer, C. J., Pantano, C., Hoffmann, S. B., Cummins, A., and Mossman, B. T. (2001) Laser-based microscopic approaches: application to cell signaling in environmental lung disease. Biotechniques 31(4), 880–894.PubMedGoogle Scholar
  2. 2.
    Cummins, A. B., Palmer, C., Mossman, B. T., and Taatjes, D. J. (2003) Persistent localization of activated extracellular signal-regulated kinases (ERK1/2) is epithelial cell-specific in an inhalation model of asbestosis. Am. J. Pathol. 162(3), 713–720.PubMedCrossRefGoogle Scholar
  3. 3.
    Lounsbury, K. M., Stern, M., Taatjes, D., Jaken, S., and Mossman, B. T. (2002) Increased localization and substrate activation of protein kinase C delta in lung epithelial cells following exposure to asbestos. Am. J. Pathol. 160(6), 1991–2000.PubMedCrossRefGoogle Scholar
  4. 4.
    Schmidt, M. H., Broll, R., Bruch, H. P., Bogler, O., and Duchrow, M. (2003) The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B. J. Pathol. 199(1), 18–27.PubMedCrossRefGoogle Scholar
  5. 5.
    Carson, F. L. (1996) Histotechnology: A Self-Instructional Text, ASCP Chicago, IL.Google Scholar
  6. 6.
    Brown, D., Lydon, J., McLaughlin, M., Stuart-Tilley, A., Tyszkowski, R., and Alper, S. (1996) Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem. Cell. Biol. 105(4), 261–267.PubMedCrossRefGoogle Scholar
  7. 7.
    Wilson, D. M., 3rd and Bianchi, C. (1999) Improved immunodetection of nuclear antigens after sodium dodecyl sulfate treatment of formaldehyde-fixed cells. J. Histochem. Cytochem. 47(8), 1095–1100.Google Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Maria Stern
    • 1
  • Douglas J. Taatjes
    • 2
  • Brooke T. Mossman
    • 1
  1. 1.Department of PathologyUniversity of VermontBurlington
  2. 2.Department of Pathology, and Microscopy Imaging CenterUniversity of VermontBurlington

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