A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part III: Reagents for Actin, Tubulin, Cellular Membranes, and Whole Cell and Cytoplasm

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

1 Molecular Probes Labeling and Detection, Life Technologies, Eugene, Oregon, 2 National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.32
DOI:  10.1002/0471142956.cy1232s67
Online Posting Date:  January, 2014
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Non‐antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole‐cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image. Curr. Protoc. Cytom. 67:12:32.1‐12:32.17. © 2014 by John Wiley & Sons, Inc.

Keywords: labeling; imaging; fluorescent dyes; cell biology; tubulin; cytoskeleton; actin; cell membrane

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Table of Contents

  • Introduction
  • Basic Protocol 1: Actin Labeling
  • Basic Protocol 2: Wheat Germ Agglutinin Conjugates for Plasma Membrane Labeling
  • Basic Protocol 3: Labeling Tubulin Microtubules with TubulinTracker Green
  • Basic Protocol 4: Labeling Whole Cells or Cytoplasm with 5(6)‐CFDA SE
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Actin Labeling

  • Cell line/type of choice
  • 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in DPBS (see recipe) or culture medium
  • 50 mM phosphate‐buffered saline (PBS), pH 7.4 (Life Technologies, cat. no. 10010)
  • 0.2% Triton X‐100 in PBS (Life Technologies, cat. no. 10010)
  • Phallotoxins (see recipe) in DPBS containing 1% to 3% (w/v BSA)
  • ProLong Gold antifade mounting medium (Life Technologies)

Basic Protocol 2: Wheat Germ Agglutinin Conjugates for Plasma Membrane Labeling

  • Cell line/type of choice
  • Appropriate culture medium, phenol red–free
  • 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in DPBS (see recipe) or culture medium (optional)
  • WGA conjugate (see recipe)
  • Hanks' balanced salt solution (HBSS; see recipe)
  • 0.2% Triton X‐100 in PBS (Life Technologies, cat. no. 10010)
  • Fluorescence microscope with filters appropriate for the conjugate dye

Basic Protocol 3: Labeling Tubulin Microtubules with TubulinTracker Green

  • Cell line/type of choice
  • Complete growth medium for selected cell line
  • 50 to 250 nm TubulinTracker Green (see recipe)
  • Physiological buffer or serum‐free and phenol red‐free cell culture medium
  • Fluorescence microscope with FITC filter set

Basic Protocol 4: Labeling Whole Cells or Cytoplasm with 5(6)‐CFDA SE

  • Cell line/type of choice
  • Complete growth medium for selected cell line
  • CFSE solution (see recipe)
  • Optimal cell culture medium for chosen cell line, serum‐free and phenol red‐free
  • 4% formaldehyde (made from methanol‐free, EM‐grade 16% formaldehyde) in
  • DPBS (see recipe) or culture medium (optional)
  • Fluorescence microscope with standard fluorescein filters
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