Cellular and Molecular Bioengineering

, Volume 4, Issue 2, pp 311–323 | Cite as

The Use of Immunofluorescent Array Tomography to Study the Three-Dimensional Microstructure of Murine Blood Vessels

  • Sanaz Saatchi
  • Nishey Wanchoo
  • Junya Azuma
  • Stephen J Smith
  • Philip S. Tsao
  • Paul G. Yock
  • Charles A. Taylor
Article

Abstract

The purpose of this study was to develop the methods needed to enable the application of Immunofluorescent Array Tomography (IAT), a novel three-dimensional (3D) microscopy technique, to murine blood vessels. The anterior and posterior regions of the infrarenal aorta of 8–10 week old C57BL6 mice were evaluated. Staining and image analysis methods were developed. Antibody selection, primary antibody concentration, co-staining with multiple primary antibodies, and the multi-cycle staining design were optimized to produce positive and specific staining of elastin (1:50 dilution), smooth muscle cell actin (SMCA, 20 μg mL−1), and collagen type I (10 μg mL−1). Non-specific interactions were limited by optimizing secondary antibody staining conditions (1:200 dilution, 30 min). SMCA and collagen type I were stained in the first cycle, elastin was stained in the second cycle, and nuclei were stained in both cycles. Algorithms were developed to quantify volume fractions of medial elastin, SMCA, and nuclei, as well as adventitial collagen type I. Elastin thickness, spacing between elastin lamellae, elastin fragmentation, media wall thickness, nuclei aspect ratio, and nuclei amount were also quantified. We have qualitatively and quantitatively characterized the 3D microstructure and cellular morphology of the anterior and posterior infrarenal murine aorta using IAT.

Keywords

Immunofluorescence microscopy Three-dimensional microstructure Murine blood vessels Infrarenal aorta Elastin Collagen type I Smooth muscle cells Volume fraction Cellular morphology 

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Copyright information

© Biomedical Engineering Society 2011

Authors and Affiliations

  • Sanaz Saatchi
    • 1
    • 5
  • Nishey Wanchoo
    • 2
  • Junya Azuma
    • 3
  • Stephen J Smith
    • 4
  • Philip S. Tsao
    • 3
  • Paul G. Yock
    • 1
  • Charles A. Taylor
    • 1
  1. 1.Department of BioengineeringStanford UniversityStanfordUSA
  2. 2.Department of Mechanical EngineeringStanford UniversityStanfordUSA
  3. 3.Department of MedicineStanford UniversityStanfordUSA
  4. 4.Department of Molecular and Cellular PhysiologyStanford UniversityStanfordUSA
  5. 5.StanfordUSA

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