Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria

Mark Terasaki1, Leslie Loew1, Jennifer Lippincott‐Schwartz2, Kristien Zaal2

1 University of Connecticut Health Center, Farmington, Connecticut, 2 National Institute of Child Health and Human Development/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.4
DOI:  10.1002/0471143030.cb0404s00
Online Posting Date:  May, 2001
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Abstract

The ability to distinguish and identify specific subcellular compartments is essential to understanding organelle function, biogenesis, and maintenance within cells and to defining protein trafficking pathways. Fluorescent dyes and/or fluorescently labeled lipid derivatives can be used to identify ER, Golgi complex, and mitochondria. Specific conditions for labeling each of these compartments are described.

     
 
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Table of Contents

  • Basic Protocol 1: Staining the Endoplasmic Reticulum in Fixed Cells
  • Alternate Protocol 1: Staining the Endoplasmic Reticulum in Living Cells
  • Basic Protocol 2: Staining the Golgi Complex in Living Cells
  • Basic Protocol 3: Staining Mitochondria
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Staining the Endoplasmic Reticulum in Fixed Cells

  Materials
  • Fixative: prepare 0.25% glutaraldehyde in PBS ( appendix 2A) from 7% to 70% commercial stock solution (store up to 1 week at 4°C)
  • Cells of interest, growing on coverslips
  • 2.5 µg/ml DiOC 6(3) working solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Silicone high‐vacuum grease
  • Small petri dish
  • Watchmaker's forceps
  • Microscope slides
  • Silicon rubber chamber (see recipe)
  • Fluorescence microscope with fluorescein filters and either 63× or 100× oil‐immersion objective (unit 4.2)
  • Additional reagents and equipment for fluorescence microscopy (unit 4.2)

Alternate Protocol 1: Staining the Endoplasmic Reticulum in Living Cells

  • 0.5 µg/ml DiOC 6(3) working solution in growth medium appropriate for cells (see recipe)

Basic Protocol 2: Staining the Golgi Complex in Living Cells

  Materials
  • Ethanol
  • 0.05% trypsin in HBSS ( appendix 2A) without calcium and magnesium
  • 1 mM ceramide fluorescent derivative working solution in ethanol (see recipe)
  • Serum‐free medium appropriate to cells
  • Cells of interest
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • 10% (w/v) defatted BSA (Sigma) in serum‐free medium appropriate to cells
  • Fluoromount G (Southern Biotechnology Associates or Electron Microscopy Sciences) or mounting medium (see recipe)
  • HEPES‐buffered culture medium appropriate to cells, pH 7.0, with 10% serum and without phenol red
  • 12‐mm diameter no. 1 round glass coverslips
  • Watchmaker's forceps
  • 10‐cm sterile tissue culture dishes
  • Hamilton syringe
  • Silicon rubber chambers (see recipe)
  • Microscope slides
  • Conventional fluorescence microscope with standard fluorescein and rhodamine filter cubes or confocal microscope with Kr/Ar laser (unit 4.2)
  • 63× (1.4 NA) or 100× (1.3 NA) oil‐immersion objective
  • Microscope air‐stream incubator (Nevtek)
  • Additional reagents and equipment for growing cells in tissue culture and trypsinization of cells (unit 1.1) and fluorescence microscopy (unit 4.2)

Basic Protocol 3: Staining Mitochondria

  Materials
  • 100 nM TMRE working solution (see recipe) in NB
  • Normal buffer (NB; see recipe)
  • No. 1, 31‐mm diameter coverslips (Bioptechs)
  • 60‐mm petri dishes
  • Forceps
  • Q tips or Kimwipes
  • Temperature‐regulated microscope chamber (Bioptechs)
  • Conventional fluorescence microscope with conventional rhodamine filter or confocal microscope (unit 4.2)
  • 40× or greater oil‐immersion objective
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Figures

Videos

Literature Cited

Literature Cited
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