Live Imaging of the Lung

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

1 Department of Pathology, University of California, San Francisco, California, 2 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, )

Support Protocol 1: Staining Lung Sections with Fluorescent Antibodies

  Materials
  • Lung sections ( 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, )

Support Protocol 2: Intratracheal and Intravascular Instillations

  • 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

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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