Use of Spectral Fluorescence Resonance Energy Transfer to Detect Nitric Oxide‐Based Signaling Events in Isolated Perfused Lung

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Fluorescence resonance energy transfer (FRET) is a fluorescence microscopy technique suitable for live cells and capable of detecting changes in the conformational state of a single protein or the distance between two interacting proteins when the proteins are conjugated with appropriate donor and acceptor fluorophores. Confocal?based spectral detection systems enable the resolution of fluorescent images by providing full spectral information for each voxel of the image without switching of optical filters. Furthermore, using calibration spectra, it is possible to unambiguously separate the cross?talk between overlapping donor and acceptor emissions. This unit describes the use of confocal?based spectral imaging of nitric oxide (NO) sensitive FRET reporters in the vasculature of the intact, isolated perfused mouse lung. This type of in situ imaging approach allows the visualization and study of temporal molecular signaling events within the appropriate physiologic microenvironment of the intact, living organ. Curr. Protocol. Cytom. 45:12.13.1?12.13.12. © 2008 by John Wiley & Sons, Inc.

Keywords: nitric oxide; fluorescence resonance energy transfer; lung; confocal microscopy

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Introduction
Strategic Planning
Basic Protocol 1: Isolating and Perfusing Mouse Lung
Basic Protocol 2: No‐Induced Protein Modifications Detected by FRET Using Spectral Confocal Microscopy
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Isolating and Perfusing Mouse Lung

Materials

Lipsosomes: 1:1 molar ratio of 1,2‐dioleoyl‐3‐trimethylammonium‐propane (DOTAP)/cholesterol (see Ma et al., , b )
Fluorescent reporter construct: e.g., FRET‐MT adenovirus or cygnet‐2 reporter (see ), containing cyan fluorescent protein (CFP; λ_ex = 440, λ_em = 480) and yellow fluorescent protein (YFP; λ_ex = 514, λ_em = 527)
C57BL/6J adult male mice (e.g., The Jackson Laboratory)
0.9% (w/v) NaCl
20 mg/ml Avertin working solution (see recipe )
Heparin
Perfusion buffer (see recipe )

Mouse restrainer (e.g., see Donovan and Brown, )
Insulin syringe (for injecting liposomes and DNA)
Peristaltic pump
Pressure transducers
Premixed normal O₂ gas cylinders (5% CO₂ , 21% O₂ , balance N₂ )
Scalpel, curved needle, silk sutures, sterile gauze
1‐cc syringe with 26‐G needle (for injecting anesthesia)
20‐G blunt metal cannula (e.g., Luer stub LS20, Instech Laboratories)
Blunt‐tipped surgical scissors
Dissecting microscope with 2× magnification
Curved‐tip hemostats
Pulmonary artery (PA) and left atrial (LA) cannulae (i.d., 1.0 mm; o.d., 1.6 mm)
Petri dish with coverslip bottom (e.g., Delta T dish, Bioptics)
Perfusate and airway lines: polyethylene PE‐50 tubing
Humidified environmental chamber: 6 × 6 × 2–in. Plexiglas chamber custom built in‐house to fit atop the Zeiss 510 META microscope stage; contains a small, 37°C heated water bath and ports for tracheal, pulmonary arterial, and atrial cannulae and a port with tubing for premixed gas tanks (see Fig. )
Spectral detection microscope system (e.g., Zeiss 510 METASystem with 40× PlanNeoFluar, 1.3‐NA, oil‐immersion objective)

Basic Protocol 2: No‐Induced Protein Modifications Detected by FRET Using Spectral Confocal Microscopy

Materials

Isolated perfused lung (IPL) preparation from mouse expressing a FRET reporter ( protocol 1 )
500 µM nitric oxide (NO) donor in perfusion buffer (see recipe ): e.g., dilute with perfusion buffer from 100 mM DETA NONOate (Alexis Biochemicals) solution in H₂ O, pH 8

Spectral detection microscope system (e.g., Zeiss 510 META with 40× PlanNeoFluar, 1.3‐NA, oil‐immersion objective) and computer with data acquisition and analysis software (e.g., Zeiss)
Argon laser (HFT 458, Zeiss)

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Figures

Figure 12.13.1 Environmental chamber housing an isolated perfused mouse lung preparation. The 6 × 6 × 2–in. Plexiglas environmental chamber was custom designed and built in‐house to fit atop the Zeiss 510 META microscope stage. It contains a small heated water bath (small open tray with water and heating coil) that keeps the interior of the chamber humidified at 37°C. Ports at either end of the chamber allow access for the tracheal (top), pulmonary arterial (left), and atrial (right) cannulae. An additional port with tubing (top left of picture) allows control of the composition of the gases within the chamber using premixed gas tanks.

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Figure 12.13.2 Nitric oxide (NO)–induced changes in FRET‐MT in intact pulmonary endothelium. Spectral laser scanning confocal imaging of small intra‐acinar arteries of the isolated perfused mouse lung showed that the addition of NO donor (500 µM DETA NONOate, left), or muscarinic agonist, activator of endothelial NO synthase (10 µM carbachol, right), to the perfusate caused an increase in the peak emission intensity of the donor (cyan, ∼485 nm) and a decrease in that of the acceptor (yellow, 525 nm) consistent with conformational changes in FRET‐MT.

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Figure 12.13.3 Selective photobleaching of FRET‐MT reporter in isolated perfused mouse lung. Acceptor photobleaching confirmed that the FRET‐MT reporter was functional in the intact pulmonary endothelium of the isolated perfused mouse lung. The graph shows the spectral report provided by the Zeiss software. Following selective photobleaching of a small region of the vessel, the donor (cyan) was unquenched, resulting in an increase in the peak emission intensity (∼485 nm) indicative of positive fluorescence resonance energy transfer (FRET). This is illustrated in the images by a color change from green to blue.

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Figure 12.13.4 Endothelial expression of FRET‐MT. Visualization was achieved via tail vein injection of DOTAP/cholesterol liposomes followed by adenovirus containing cDNA for FRET‐MT. Expression in the endothelium was confirmed by (A ) colocalization of FRET‐MT and PECAM (platelet/endothelial cell adhesion molecule) in fixed tissue and (B ) perfusion of the isolated lung preparation with Cy3‐labeled albumin to visualize the pulmonary vascular network.

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Literature Cited

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