Cell Imaging Techniques pp 85-95

Part of the Methods in Molecular Biology book series (MIMB, volume 931)

Multifluorescence Confocal Microscopy: Application for a Quantitative Analysis of Hemostatic Proteins in Human Venous Valves

  • Winifred E. Trotman
  • Douglas J. Taatjes
  • Edwin G. Bovill


Confocal laser scanning microscopy is commonly used to visualize and quantify protein expression. Visualization of the expression of multiple proteins in the same region via multifluorescence allows for the analysis of differential protein expression. The defining step of multifluorescence labeling is the selection of primary antibodies from different host species. In addition, species-appropriate secondary antibodies must also be conjugated to different fluorophores so that each protein can be visualized in separate channels. Quantitative analysis of proteins labeled via multifluorescence can be used to compare relative changes in protein expression. Multifluoresecence labeling and analysis of fluorescence intensity within and among human venous specimens, for example, allowed us to determine that the anticoagulant phenotype of the venous valve is defined not by increased anticoagulant expression, but instead by significantly decreased procoagulant protein expression (Blood 114:1276–1279, 2009 and Histochem Cell Biol 135:141–152, 2011).

Key words

Multifluorescence Confocal laser scanning microscopy Immunofluorescence Fluorescence intensity Quantitative analysis 


  1. 1.
    Brooks EG, Trotman WE, Wadsworth MP, Taatjes DJ, Evans MF, Ittleman FP, Callas PW, Esmon CT, Bovill EG (2009) Valves of the deep venous system: an overlooked risk factor. Blood 114:1276–1279PubMedCrossRefGoogle Scholar
  2. 2.
    Trotman WE, Taatjes DJ, Callas PW, Bovill EG (2011) The endothelial microenvironment in the venous valvular sinus: thromboresistance trends and inter-individual variation. Histochem Cell Biol 135:141–152PubMedCrossRefGoogle Scholar
  3. 3.
    Lichtman JW, Conchello J-A (2005) Fluorescence microscopy. Nat Methods 2:910–919PubMedCrossRefGoogle Scholar
  4. 4.
    Conchello J-A, Lichtman JW (2005) Optical sectioning microscopy. Nat Methods 2:920–931PubMedCrossRefGoogle Scholar
  5. 5.
    Amos WB, White JG (2003) How the confocal laser scanning microscope entered biological research. Biol Cell 95:335–342PubMedCrossRefGoogle Scholar
  6. 6.
    Stern M, Taatjes DJ, Mossman BT (2005) Multifluorescence labeling techniques and confocal laser scanning microscopy on lung tissue. Methods Mol Biol 319:67–76CrossRefGoogle Scholar
  7. 7.
    Sedgewick J (2007) Guidelines for a specific type of images. Scientific imaging with Photoshop: methods, measurements and output. New Riders, Berkeley, pp 44–74Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Winifred E. Trotman
    • 1
  • Douglas J. Taatjes
    • 2
  • Edwin G. Bovill
    • 1
  1. 1.Department of Pathology, College of MedicineUniversity of VermontBurlingtonUSA
  2. 2.Department of Pathology and Microscopy Imaging Center, College of MedicineUniversity of VermontBurlingtonUSA

Personalised recommendations