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 1: 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 1: 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 9.9.1   MaterialsProperties of Selected Vital Dyes

    Dye Dye excitation/emission (nm) Excitation laser Working concentration Suggested emission filter Quality of staining with ex vivo tissue imaging a
    10‐n‐Nonyl Acridine Orange (NAO) 500/525 488 nm 400 nM 505‐530 BP or 505‐570 BP ++
    Calcein AM 490/525 488 nm 5 µM 505‐530 BP or 505‐570 BP ++
    Cell Rox Deep Red 644/665 647 nm 5 µM 650 LP +++
    Cell Tracker Blue 353/466 405 nm 5 µM 420‐480 BP +++
    Draq5 650/680 647 nm 5 µM 650 LP +++
    Ethidium homodimer‐1 (EthD‐1) 528/617 514 nm 1 µM 560‐615 BP ++
    GFP (as LC3‐GFP) 470/514 488 nm n/a 505‐530 BP or 505‐570 BP +++
    H 2CMXRos 543/580 543 nm 200 nM 560‐615 BP ++
    H 2DCFDA 495/525 488 nm 5 µM 505‐530 BP or 505‐570 BP +
    Hoechst 33342 350/460 405 nm 5 µg/ml 420‐480 BP +++
    JC‐1 Monomer ‐ 485/530 488 nm 10 µg/ml 505‐530 BP
    Aggregate ‐ 535/590 543 nm 560‐615 BP
    LysoSensor Blue DND‐167 373/425 405 nm 500 nM 420‐480 BP +
    MCB 394/490 405 nm 60 µM 420‐480 BP ++
    MitoPY1 543/560 543 nm 5 µM 560‐615 BP +++
    MitoSox Non‐specific product 510/590 514 nm 5 µM 560‐615 BP ++
    Superoxide specific product 396/590 405 nm 5 µM 560‐615 BP ++
    MitoTracker Deep Red 640/662 647 nm 200 nM 650 LP +++
    MitoTracker Green 490/516 488 nm 200 nM Mitochondrial mass +
    Sytox Green 504/523 488 nm 100 nM 505‐530 BP +++
    TMRE 545/590 543 nm 200 nM 560‐615 BP +++
    Vybrant Ruby 638/686 647 nm 5 µM 650 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

  • 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

  • 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

  • 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

  • A mix of dyes chosen based on parameters of interest, spectral compatibility, available lasers and filter sets, etc. (see protocol 4 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

  • A mix of dyes chosen based on parameters of interest, spectral compatibility, available lasers and filter sets, etc. (see protocol 4 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

  • 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 protocol 4 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

Videos

Literature Cited

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Internet Resources
   http://rsbweb.nih.gov/nih‐image/
  NIH ImageJ software.
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