Live Imaging of the Lung

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Videos
Literature Cited

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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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

Figure 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.

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Figure 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.

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Figure 12.28.3 Staining of Lyve‐1 and CD31 in lung slices. Lung sections stained as in show Lyve‐1 (blue) and CD31 (red).

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Figure 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., ).

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Videos

Neutrophil and macrophage motility in lung sections.

Duration: 0:08

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Simultaneous imaging of neutrophil and bead velocities in the mouse lung.

Duration: 0:33

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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.