Skip to main content
SpringerLink
Log in

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

  • Published:
Cellular and Molecular Bioengineering Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Ailawadi, G., J. L. Eliason, and G. R. Upchurch, Jr. Current concepts in the pathogenesis of abdominal aortic aneurysm. J. Vasc. Surg. 38(3):584–588, 2003.

    Article  Google Scholar 

  2. Ailawadi, G., C. W. Moehle, H. Pei, S. P. Walton, Z. Yang, I. L. Kron, C. L. Lau, and G. K. Owens. Smooth muscle phenotypic modulation is an early event in aortic aneurysms. J. Thorac. Cardiovasc. Surg. 138(6):1392–1399, 2009.

    Article  Google Scholar 

  3. Berry, C. L., T. Looker, and J. Germain. The growth and development of the rat aorta. I. Morphological aspects. J. Anat. 113(Pt 1):1–16, 1972.

    Google Scholar 

  4. Berry, C. L., J. A. Sosa-Melgarejo, and S. E. Greenwald. The relationship between wall tension, lamellar thickness, and intercellular junctions in the fetal and adult aorta: its relevance to the pathology of dissecting aneurysm. J. Pathol. 169(1):15–20, 1993.

    Article  Google Scholar 

  5. Brooke, B. S., A. Bayes-Genis, and D. Y. Li. New insights into elastin and vascular disease. Trends Cardiovasc. Med. 13(5):176–181, 2003.

    Article  Google Scholar 

  6. Bunton, T. E., N. J. Biery, L. Myers, B. Gayraud, F. Ramirez, and H. C. Dietz. Phenotypic alteration of vascular smooth muscle cells precedes elastolysis in a mouse model of Marfan syndrome. Circ. Res. 88(1):37–43, 2001.

    Google Scholar 

  7. Cho, B. S., K. J. Roelofs, J. W. Ford, P. K. Henke, and G. R. Upchurch, Jr. Decreased collagen and increased matrix metalloproteinase-13 in experimental abdominal aortic aneurysms in males compared with females. Surgery 147(2):258–267, 2010.

    Article  Google Scholar 

  8. Clark, J. M., and S. Glagov. Transmural organization of the arterial media. The lamellar unit revisited. Arteriosclerosis 5(1):19–34, 1985.

    Google Scholar 

  9. Dai, D., Y. H. Ding, D. A. Lewis, and D. F. Kallmes. A proposed ordinal scale for grading histology in elastase-induced, saccular aneurysms. AJNR Am. J. Neuroradiol. 27(1):132–138, 2006.

    Google Scholar 

  10. Davies, J. Q., and S. Gordon. Isolation and culture of murine macrophages. Methods Mol. Biol. 290:91–103, 2005.

    Google Scholar 

  11. Dingemans, K. P., P. Teeling, J. H. Lagendijk, and A. E. Becker. Extracellular matrix of the human aortic media: an ultrastructural histochemical and immunohistochemical study of the adult aortic media. Anat. Rec. 258(1):1–14, 2000.

    Article  Google Scholar 

  12. Hao, H., G. Gabbiani, and M. L. Bochaton-Piallat. Arterial smooth muscle cell heterogeneity: implications for atherosclerosis and restenosis development. Arterioscler. Thromb. Vasc. Biol. 23(9):1510–1520, 2003.

    Article  Google Scholar 

  13. Lopez-Candales, A., D. R. Holmes, S. Liao, M. J. Scott, S. A. Wickline, and R. W. Thompson. Decreased vascular smooth muscle cell density in medial degeneration of human abdominal aortic aneurysms. Am. J. Pathol. 150(3):993–1007, 1997.

    Google Scholar 

  14. Micheva, K. D., and S. J Smith. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55(1):25–36, 2007.

    Google Scholar 

  15. Ntziachristos, V. Going deeper than microscopy: the optical imaging frontier in biology. Nat. Methods 7(8):603–614, 2010.

    Article  Google Scholar 

  16. O’Connell, M. K., S. Murthy, S. Phan, C. Xu, J. Buchanan, R. Spilker, R. L. Dalman, C. K. Zarins, W. Denk, and C. A. Taylor. The three-dimensional micro- and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imaging. Matrix Biol. 27(3):171–181, 2008.

    Article  Google Scholar 

  17. Thompson, R. W., J. A. Curci, T. L. Ennis, D. Mao, M. B. Pagano, and C. T. Pham. Pathophysiology of abdominal aortic aneurysms: insights from the elastase-induced model in mice with different genetic backgrounds. Ann. N.Y. Acad. Sci. 1085:59–73, 2006.

    Article  Google Scholar 

  18. Vanhecke, D., S. Asano, Z. Kochovski, R. Fernandez-Busnadiego, N. Schrod, W. Baumeister, and V. Lucic. Cryo-electron tomography: methodology, developments and biological applications. J Microsc. [Epub ahead of print], 2010.

  19. Wasano, K., and T. Yamamoto. Tridimensional architecture of elastic tissue in the rat aorta and femoral artery—a scanning electron microscope study. J. Electron. Microsc. (Tokyo) 32(1):33–44, 1983.

    Google Scholar 

  20. Wolinsky, H., and S. Glagov. A lamellar unit of aortic medial structure and function in mammals. Circ. Res. 20(1):99–111, 1967.

    Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge all members of the Smith Laboratory at Stanford University, especially Brad L. Busse, Dr. Kristina D. Micheva, Dr. Nancy A. O’Rourke, and Dr. Gordon X. Wang, for their technical expertise in IAT, support in developing the presented methods, and review of the manuscript. We also acknowledge Dr. Joan M. Greve for her encouragement, support, and critical review of the manuscript. The authors also gratefully acknowledge all staff members of the Cell Sciences Imaging Facility at Stanford University, especially Jon W. Mulholland and Ibanri Phanwar, for technical guidance and all array generation. We would also like to acknowledge Dr. Andrew J. Connolly in the Pathology Department at Stanford University for his expertise in vascular histology and staining. S. Saatchi was supported by a Stanford University Bio-X Graduate Student Fellowship sponsored by Amgen Inc. This research was funded in part by the NIH (1P50HL083800). The authors do not have any conflicts of interest.

Author information

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Sanaz Saatchi, Paul G. Yock & Charles A. Taylor

  2. Department of Mechanical Engineering, Stanford University, Stanford, CA, USA

    Nishey Wanchoo

  3. Department of Medicine, Stanford University, Stanford, CA, USA

    Junya Azuma & Philip S. Tsao

  4. Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA

    Stephen J Smith

  5. 318 Campus Drive, Room E350, Stanford, CA, 94305, USA

    Sanaz Saatchi

Authors
  1. Sanaz Saatchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nishey Wanchoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Junya Azuma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephen J Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Philip S. Tsao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Paul G. Yock
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Charles A. Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sanaz Saatchi.

Additional information

Associate Editor Andrew D. McCulloch oversaw the review of this article.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Saatchi, S., Wanchoo, N., Azuma, J. et al. The Use of Immunofluorescent Array Tomography to Study the Three-Dimensional Microstructure of Murine Blood Vessels. Cel. Mol. Bioeng. 4, 311–323 (2011). https://doi.org/10.1007/s12195-011-0165-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12195-011-0165-z

Keywords

Search

Navigation