Cell Imaging Techniques

Volume 931 of the series Methods in Molecular Biology pp 85-95


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

  • Winifred E. TrotmanAffiliated withDepartment of Pathology, College of Medicine, University of Vermont
  • , Douglas J. TaatjesAffiliated withDepartment of Pathology and Microscopy Imaging Center, College of Medicine, University of Vermont Email author 
  • , Edwin G. BovillAffiliated withDepartment of Pathology, College of Medicine, University of Vermont

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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