A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part II: Reagents for Non‐Vesicular Organelles

Jason A. Kilgore1, Nick J. Dolman1, Michael W. Davidson2

1 Life Technologies, Eugene, Oregon, 2 Florida State University, Tallahassee, Florida
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.31
DOI:  10.1002/0471142956.cy1231s66
Online Posting Date:  October, 2013
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Abstract

A wide range of fluorescent dyes and reagents exist for labeling organelles in live and fixed cells. Choosing between them can sometimes be confusing, and optimization for many of them can be challenging. Presented here is a discussion on the commercially‐available reagents that have shown the most promise for each organelle of interest, including endoplasmic reticulum/nuclear membrane, Golgi apparatus, mitochondria, nucleoli, and nuclei, with an emphasis on localization of these structures for microscopy. Included is a featured reagent for each structure with a recommended protocol, troubleshooting guide, and example image. Curr. Protoc. Cytom. 66:12.31.1‐12.34.24. © 2013 by John Wiley & Sons, Inc.

Keywords: labeling; imaging; fluorescent dyes; organelles; cell biology

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Endoplasmic Reticulum and Nuclear Membrane Labeling Using ER‐Tracker Reagents
  • Basic Protocol 2: Labeling Golgi Apparatus Using Dye‐Labeled Ceramides
  • Basic Protocol 3: Labeling Mitochondria Using MitoTracker Red CMXRos
  • Basic Protocol 4: Labeling Nucleoli Using Syto RNASelect Green
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Endoplasmic Reticulum and Nuclear Membrane Labeling Using ER‐Tracker Reagents

  Materials
  • Cell line/type of choice
  • Appropriate culture medium, phenol red–free
  • ER‐Tracker reagent (see recipe)
  • Hank's balanced salt solution (HBSS; see recipe) is preferred, but any physiological buffer or phenol red–free medium will work
  • 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in DPBS (see recipe) or culture medium (optional)
  • 0.2% Triton X‐100 in PBS (Life Technologies, cat. no. 10010)
  • ProLong Gold antifade mounting medium (Life Technologies; optional)

Basic Protocol 2: Labeling Golgi Apparatus Using Dye‐Labeled Ceramides

  Materials
  • Cell line/type of choice
  • Appropriate culture medium, phenol red–free
  • Hanks’ balanced salt solution (HBSS, see recipe; DPBS, see recipe, may also be used)
  • Fluorescent ceramide analog conjugated to BSA (see recipe)
  • Glass‐bottom Petri dishes, coverslips, or multi‐well plates appropriate for cell line
  • Fluorescence microscope with filter sets for NBD, BODIPY Fl, or BODIPY TR

Basic Protocol 3: Labeling Mitochondria Using MitoTracker Red CMXRos

  Materials
  • Cell line/type of choice
  • Appropriate culture medium, phenol red–free
  • Test compounds (or other test conditions) p‐(trifluoromethoxy)phenylhydrazone (FCCP; Sigma‐Aldrich, cat. no. F2920), carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; Sigma‐Aldrich, cat. no. C2759), or comparable protonophore MitoTracker Red CMXRos (see recipe)
  • 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in HBSS (see recipe)
  • 50 mM phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010)
  • 0.2% Triton X‐100 in HBSS
  • Fluorescence microscope with TRITC or Alexa Fluor 568 filter set

Basic Protocol 4: Labeling Nucleoli Using Syto RNASelect Green

  Materials
  • Cell line/type of choice
  • 100% methanol, ice‐cold (optional)
  • Appropriate cell culture medium or suitable live‐cell buffer, phenol red‐free
  • 500 nM SYTO RNASelect Green working solution (see recipe)
  • Curing mounting medium, e.g., ProLong Gold (Life Technologies)
  • Fluorescence microscope with FITC filter set
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Figures

Videos

Literature Cited

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