Ex Vivo Imaging of Excised Tissue Using Vital Dyes and Confocal Microscopy

Simon Johnson1, Peter Rabinovitch1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.39
DOI:  10.1002/0471142956.cy0939s61
Online Posting Date:  July, 2012
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Abstract

Vital dyes routinely used for staining cultured cells can also be used to stain and image live tissue slices ex vivo. Staining tissue with vital dyes allows researchers to collect structural and functional data simultaneously and can be used for qualitative or quantitative fluorescent image collection. The protocols presented here are useful for structural and functional analysis of viable properties of cells in intact tissue slices, allowing for the collection of data in a structurally relevant environment. With these protocols, vital dyes can be applied as a research tool to disease processes and properties of tissue not amenable to cell culture–based studies. Curr. Protoc. Cytom. 61:9.39.1‐9.39.18. © 2012 by John Wiley & Sons, Inc.

Keywords: vital dyes; confocal microscopy; live cell staining; tissue imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol: Staining and Imaging Mitochondria, Nuclei, and GFP in Live Tissue Slices Ex Vivo
  • Alternate Protocol 1: Assessing Cell Viability in Live Tissue Slices Ex Vivo Using Ethidium Homodimer‐1 and Calcein AM
  • Alternate Protocol 2: Assessing Cell Viability in Live Tissue Slices Ex Vivo Using Sytox Green and Tetramethylrhodamine Ethyl Ester
  • Alternate Protocol 3: Generalized Method for Tissue Slice Staining Ex Vivo
  • Alternate Protocol 4: Depth‐Dependent Quantitative Data Collection
  • Alternate Protocol 5: Depth‐Independent Quantitative Data Collection
  • Alternate Protocol 6: Staining of Tissues Using Dyes Dependent on Active Physiological Functions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Staining and Imaging Mitochondria, Nuclei, and GFP in Live Tissue Slices Ex Vivo

 Materials
  • Freshly excised tissue from an animal expressing GFP
  • Excision and incubation buffer (1× HBSS with additives, see recipe)
  • Ice
  • 1 mg/ml Hoechst 33342 in 1× PBS (see recipe)
  • 200 µM MitoTracker Deep Red in DMSO (see recipe)
  • Hanks balanced salt solution (HBSS)
  • Immersion oil, optional
  • Weigh boat or small dish on ice
  • Recommended for mouse tissue: mouse heart or brain slicing block (Zivic, cat. nos. HSMS001.1, BSMS001.1, or BSMS001.2)
  • Double‐edged razor blade (we prefer Fisher, cat. no. NC9873485)
  • 12‐well tissue culture plates or equivalent size polystyrene plates
  • Forceps
  • Orbital shaker capable of rotating at up to 100 to 150 rpm with ice bucket securely on top (or an orbital shaker in a walk‐ in cold room)
  • 2‐well chambered coverglass slides (Thermo, cat. no. 155379)
  • Aluminum foil folded to rigid square of ∼2 cm × 2 cm × 8‐folds thickness
  • 1.8‐ml glass sample vial (Thermo, cat. no. 03‐339‐25A) or an object of similar size and a mass of 3 to 5 g, suitable for gently compressing tissue
  • Confocal microscope with desired objectives and with lasers and filters for DAPI, FITC, and far‐red excitation and emission (see Table 9.39.1 for suggested laser and filter sets)
     
    Table 9.39.1 Properties of Selected Vital Dyes
    DyeDye excitation/emission (nm)Excitation laserWorking concentrationSuggested emission filterQuality of staining with ex vivo tissue imaginga
    10‐n‐Nonyl Acridine Orange (NAO)500/525488 nm400 nM505‐530 BP or 505‐570 BP++
    Calcein AM490/525488 nm5 µM505‐530 BP or 505‐570 BP++
    Cell Rox Deep Red644/665647 nm5 µM650 LP+++
    Cell Tracker Blue353/466405 nm5 µM420‐480 BP+++
    Draq5650/680647 nm5 µM650 LP+++
    Ethidium homodimer‐1 (EthD‐1)528/617514 nm1 µM560‐615 BP++
    GFP (as LC3‐GFP)470/514488 nmn/a505‐530 BP or 505‐570 BP+++
    H2CMXRos543/580543 nm200 nM560‐615 BP++
    H2DCFDA495/525488 nm5 µM505‐530 BP or 505‐570 BP+
    Hoechst 33342350/460405 nm5 µg/ml420‐480 BP+++
    JC‐1Monomer ‐ 485/530488 nm10 µg/ml505‐530 BP
    Aggregate ‐ 535/590543 nm560‐615 BP
    LysoSensor Blue DND‐167373/425405 nm500 nM420‐480 BP+
    MCB394/490405 nm60 µM420‐480 BP++
    MitoPY1543/560543 nm5 µM560‐615 BP+++
    MitoSoxNon‐specific product 510/590514 nm5 µM560‐615 BP++
    Superoxide specific product 396/590405 nm5 µM560‐615 BP++
    MitoTracker Deep Red640/662647 nm200 nM650 LP+++
    MitoTracker Green490/516488 nm200 nMMitochondrial mass+
    Sytox Green504/523488 nm100 nM505‐530 BP+++
    TMRE545/590543 nm200 nM560‐615 BP+++
    Vybrant Ruby638/686647 nm5 µM650 LP+++
     aQuality of staining refers to the general opinion of the authors of this paper of the utility of these dyes using this method. Specificity, brightness, ability to permeate tissue, and photostability are all included in this determination. The highest quality dyes are designated with +++, whereas dyes that were unfavorable are designated with +.

Alternate Protocol 1: Assessing Cell Viability in Live Tissue Slices Ex Vivo Using Ethidium Homodimer‐1 and Calcein AM

 Additional Materials (also see Basic Protocol)
  • 2 mM EthD‐1 in DMSO (Sigma, cat. no. E1903)
  • 4 mM Calcein AM in DMSO (Sigma, cat. no. C1359)

Alternate Protocol 2: Assessing Cell Viability in Live Tissue Slices Ex Vivo Using Sytox Green and Tetramethylrhodamine Ethyl Ester

 Additional Materials (also see Basic Protocol)
  • 5 mM Sytox Green in DMSO (Invitrogen, cat. no. S7020)
  • 200 µM TMRE in DMSO (see recipe)

Alternate Protocol 3: Generalized Method for Tissue Slice Staining Ex Vivo

 Additional Materials (also see Basic Protocol)
  • A mix of dyes chosen based on parameters of interest, spectral compatibility, available lasers, and filter sets, etc. (see protocol steps for details on selection of dye sets and Table 9.39.1 for the properties of dyes tested with this technique)

Alternate Protocol 4: Depth‐Dependent Quantitative Data Collection

 Additional Materials (also see Basic Protocol)
  • A mix of dyes chosen based on parameters of interest, spectral compatibility, available lasers and filter sets, etc. (see Alternate Protocol 3 for details on selection of dye sets and Table 9.39.1 for the properties of dyes tested with this technique)

Alternate Protocol 5: Depth‐Independent Quantitative Data Collection

 Additional Materials (also see Basic Protocol)
  • A mix of dyes chosen based on parameters of interest, spectral compatibility, available lasers and filter sets, etc. (see Alternate Protocol 3 for details on selection of dye sets and Table 9.39.1 for the properties of dyes tested with this technique)

Alternate Protocol 6: Staining of Tissues Using Dyes Dependent on Active Physiological Functions

 Additional Materials (also see Basic Protocol)
  • Staining buffer of choice (see recipe for generic staining buffer)
  • A mix of dyes chosen based on parameters of interest, spectral compatibility, available lasers and filter sets, etc. (see Alternate Protocol 3 for details on selection of dye sets and Table 9.39.1 for the properties of dyes tested with this technique)
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Figures

  •  FigureFigure 9.39.1 Sample images collected using vital dyes and ex vivo imaging sample images of tissues stained with vital dyes using the protocols in this unit. (A), (B), and (F) show, in order, mouse slow twitch and fast twitch skeletal muscle fibers and mouse optic nerve stained with TMRE and Hoechst 33342. Panel (C) is a sample image of human colon biopsy sample stained with TMRE and Cell Tracker Blue. (D) and (E) are mouse cerebellum and liver expressing LC3‐GFP stained with TMRE and Hoechst 33343 (as in Basic Protocol). Tissue, cellular, and subcellular structures are easily distinguished using these methods.
    View Image
  •  FigureFigure 9.39.2 Tissue viability analysis with depth using ethidium homodimer‐1 and Calcein AM or Sytox Green and tetramethylrhodamine ethyl ester tissue viability assessed using the staining methods in Alternate Protocols 1 and 2. (A) Liver tissue stained with Calcein AM and EthD‐1. At the cut surface a small percentage of cells show lost membrane integrity by EthD‐1 positivity (A1), but the frequency of EthD‐1 positive cells quickly drops to nearly zero with depth. (B) Cardiac tissue stained with Calcein AM and EthD‐1. Tissue cut in parallel with the cardiac fibers has few EthD‐1 positive cells (B1‐B2), demonstrating almost complete viability even just interior of the cut surface. Tissue cut transverse to the fibers has a slightly increased rate of membrane integrity loss as seen by a higher rate of EthD‐1 positive staining (B1, B3). (C) Renal glomeruli stained with Sytox Green and TMRE. The rate of Sytox positive cells is highest near the surface and steadily drops with depth. By image analysis it is appears that the bulk of the Sytox positive cells do not contribute to the structures of interest, the glomeruli and renal tubules, which appear completely viable by Sytox staining. TMRE positive staining shows that the mitochondria in these cells are functional, further indicating the cells are viable.
    View Image
  •  FigureFigure 9.39.3 Quantitative data collection and analysis. (A) Schematic of the collection of a z‐stack of images. A z‐scan of the region of interest allows the first optical slice to be set at the cut surface (the tissue‐coverglass interface) and the distance between optical slices to be specified. The first image in each stack, the cut surface image, is referred to as the z = 0 slice. This optical slice is collected to allow for orientation of the other slices, but is not included in any analyses. (B1). Multiple z‐stacks of images in a single sample are analyzed by depth. Average values for a given fluorophore across multiple depths are plotted and the approximate rate of signal loss is determined, as shown by (a) and (b). The calculated rate of signal loss is used to normalize the data by depth so that the data from all images of a given fluorophore in an optical stack, and across multiple stacks, can be directly averaged. The result of signal loss compensation is shown in (B2). For the sake of presentation, each individual fluorophore is normalized to the relative z = 1 signal. Each dye or fluorophore must be empirically examined. (B1) and (B3) show that fluorophore signal loss is dye dependent.
    View Image

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 Internet Resources
    http://rsbweb.nih.gov/nih‐image/

NIH ImageJ software.

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