Live Imaging of the Lung

Emily E. Thornton1, Matthew F. Krummel1, Mark R. Looney2,3

1 Department of Pathology, University of California, San Francisco, California, 2 Department of Medicine, University of California, San Francisco, California, 3 Department of Laboratory Medicine, University of California, San Francisco, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.28
DOI:  10.1002/0471142956.cy1228s60
Online Posting Date:  April, 2012
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Abstract

Live imaging is critical to determining the dynamics and spatial interactions of cells within the tissue environment. In the lung, this has proven to be difficult due to the motion incurred by ventilation and cardiac contractions. In this chapter, we report protocols for imaging ex vivo live lung slices and the intact mouse lung. Curr. Protoc. Cytom. 60:12.28.1‐12.28.12. © 2012 by John Wiley & Sons, Inc.

Keywords: intravital imaging; lung imaging; lung slices; two‐photon microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Live Imaging of Lung Slices
  • Support Protocol 1: Staining Lung Sections with Fluorescent Antibodies
  • Basic Protocol 2: Live Imaging in the Mouse Lung
  • Support Protocol 2: Intratracheal and Intravascular Instillations
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Live Imaging of Lung Slices

 Materials
  • Transgenic mouse of interest
  • Anesthetic: e.g., 2.5% (w/v) Avertin
  • 70% (v/v) ethanol
  • 2% (w/v) low‐melting‐temperature agarose (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • Vetbond (3M)
  • RPMI 1640 medium without phenol red
  • Dissection board
  • Dissection instruments
  • Suture: 3–0 silk
  • Plastic sheath from 18‐G catheter (Exel Safelet Cath, Fisher Scientific)
  • 1‐ml syringe without a needle
  • Vibratome
  • Plastic cover slips (Fisher)
  • Microscope
  • Perfusion system: peristaltic pump, in‐line heater, and heated imaging chamber (Warner Instruments)
  • Additional reagents and equipment for intraperitoneal injection of mice (Donovan and Brown, 2006)

Support Protocol 1: Staining Lung Sections with Fluorescent Antibodies

 Materials
  • Lung sections (Basic Protocol 1, step 15)
  • RPMI 1640 medium without phenol red
  • Primary antibodies: Lyve‐1, unconjugated (R&D) and PE‐conjugated CD31 (BioLegend)
  • Secondary antibody: anti‐goat Dylight 649 for Lyve‐1 primary (Jackson ImmunoResearch)
  • 24‐well cell culture plate
  • Shaking platform

Basic Protocol 2: Live Imaging in the Mouse Lung

 Materials
  • Mice (procedure is best performed in adult mice >20 g in body weight)
  • Anesthetics: ketamine, xylazine, isoflurane
  • Compressible gases (21%, 100% oxygen)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Alcohol swabs
  • Two‐photon microscope with motorized and heated stage
  • Rectal thermometer
  • Adhesive tape
  • Suture: 3–0 silk
  • Fiber optic illuminator (Cole Parmer) for surgery
  • Surgical tools for tracheotomy and thoracotomy
  • PE‐90 tubing (Intramedic)
  • Mechanical ventilator (e.g., Kent Scientific or Harvard Apparatus)
  • Isoflurane vaporizer (Molecular Imaging Products, cat. no. AS‐01‐0007; http://www.mipcompany.com/)
  • Customized thoracic suction window (see annotation to step 14, below)
  • Micromanipulator (Thorlabs; http://www.thorlabs.com)
  • 12‐mm glass coverslips
  • Suction regulator and tubing
  • Additional reagents and equipment for intraperitoneal injection of mice (Donovan and Brown, 2006)

Support Protocol 2: Intratracheal and Intravascular Instillations

 Additional Materials (also see Basic Protocols 1 and 2)
  • Dextran‐conjugated dyes (Invitrogen; also see recipe)
  • Fluorescent dyes or microbeads (Invitrogen; also see recipe)
  • PE‐10 tubing (Intramedic)
  • 30‐G needle (or the smallest you can find)
  • Syringes (0.5 to 1 ml sizes)
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Figures

  •  FigureFigure 12.28.1 Protocol for preparing lungs for slice imaging. (A) Catheter inserted in the trachea is stabilized with suture. (B) Lungs filled with 1 ml low melting temperature agarose. (C) Left lobe mounted on a Vibtratome block with Vetbond. (D) 300‐µm section immobilized on a plastic coverslip.
    View Image
  •  FigureFigure 12.28.2 Motility of c‐fms‐EGFP+ cells in lung sections. (A) Actin‐CFP c‐fms‐EGFP lung slice shows motility of cells within the tissue with overlaid tracks. (B) Quantification of c‐fms‐EGFP cells shows increased motility near the airway.
    View Image
  •  FigureFigure 12.28.3 Staining of Lyve‐1 and CD31 in lung slices. Lung sections stained as in Support Protocol 1 show Lyve‐1 (blue) and CD31 (red).
    View Image
  •  FigureFigure 12.28.4 Surgical preparation for lung intravital microscopy. (A) Anterior and posterior views of the thoracic suction window fitted with a coverslip. (B) Side‐view rendering of the suction window showing suction chamber, cover slip (green arrows), and vacuum flows (blue arrows near tissue, red arrows toward suction regulator). (C) Surgical preparation of left thorax with exposed left lung. (D) Suction window in situ. Scale bars, 5 mm (B), 10 mm (C,D) (Looney et al., 2011).
    View Image

Literature Cited

Literature Cited
    Bergner, A. and Sanderson, M.J. 2002. Acetylcholine‐induced calcium signaling and contraction of airway smooth muscle cells in lung slices. J. Gen. Physiol. 119:187‐198.
    Dandurand, R.J., Wang, C.G., Phillips, N.C., and Eidelman, D.H. 1993. Responsiveness of individual airways to methacholine in adult rat lung explants. J. Appl. Physiol. 75:364‐372.
    Donovan, J. and Brown, P. 2006. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
    Hasegawa, A., Hayashi, K., Kishimoto, H., Yang, M., Tofukuji, S., Suzuki, K., Nakajima, H., Hoffman, R.M., Shirai, M., and Nakayama, T. 2010. Color‐coded real‐time cellular imaging of lung T‐lymphocyte accumulation and focus formation in a mouse asthma model. J. Allergy Clin. Immunol. 125:461‐468.
    Kiefmann, R., Rifkind, J.M., Nagababu, E., and Bhattacharya, J. 2008. Red blood cells induce hypoxic lung inflammation. Blood 111:5205‐5214.
    Kreisel, D., Nava, R.G., Li, W., Zinselmeyer, B.H., Wang, B., Lai, J., Pless, R., Gelman, A.E., Krupnick, A.S., and Miller, M.J. 2010. In vivo two‐photon imaging reveals monocyte‐dependent neutrophil extravasation during pulmonary inflammation. Proc. Natl. Acad. Sci. U.S.A. 107:18073‐18078.
    Kuebler, W.M., Ying, X., Singh, B., Issekutz, A.C., and Bhattacharya, J. 1999. Pressure is proinflammatory in lung venular capillaries. J. Clin. Invest. 104:495‐502.
    Looney, M.R., Thornton, E.E., Sen, D., Lamm, W.J., Glenny, R.W., and Krummel, M.F. 2011. Stabilized imaging of immune surveillance in the mouse lung. Nat. Methods 8:91‐96.
    Miller, M.J., Wei, S.H., Parker, I., and Cahalan, M.D. 2002. Two‐photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296:1869‐1873.
    Su, X., Looney, M., Robriquet, L., Fang, X., and Matthay, M.A. 2004. Direct visual instillation as a method for efficient delivery of fluid into the distal airspaces of anesthetized mice. Exp. Lung Res. 30:479‐483.
    Tabuchi, A., Mertens, M., Kuppe, H., Pries, A.R., and Kuebler, W.M. 2008. Intravital microscopy of the murine pulmonary microcirculation. J. Appl. Physiol. 104:338‐346.
    Wagner, W.W. Jr. 1996. Pulmonary microcirculatory observations in vivo under physiological conditions. J. Appl. Physiol. 26:375‐377.
 Key References
    Looney et al., 2011. See above.

This reference describes in detail the methods needed for two‐photon, intravital microscopy in the lung.

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