Point by Point: An Introductory Guide to Sample Preparation for Single‐Molecule, Super‐Resolution Fluorescence Microscopy

Aaron R. Halpern1, Marco D. Howard1, Joshua C. Vaughan2

1 Department of Chemistry, University of Washington, Seattle, Washington, 2 Department of Physiology and Biophysics, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140241
Online Posting Date:  June, 2015
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Abstract

Single‐molecule, localization‐based, super resolution microscopy is able to reveal detailed subcellular structures and protein distributions below the classical ∼250‐nm diffraction limit of light, but utilizing this technique effectively requires a combination of careful sample preparation, data acquisition, and data analysis, which can be daunting to novice researchers. In this protocol, detailed instructions on preparation of robust reference samples for super‐resolution microscopy, including the cytoskeleton (microtubules), membrane‐bound organelles (peroxisomes), and scaffold proteins (clathrin), are provided. These samples also constitute a representative subset of imaging targets of interest to biological researchers and highlight the differences and similarities in sample preparation. © 2015 by John Wiley & Sons, Inc.

Keywords: super‐resolution microscopy; STORM; PALM; microtubules; peroxisomes; clathrin

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunostaining of Cultured Cells
  • Support Protocol 1: Secondary Antibody Labeling with NHS Fluorophores
  • Support Protocol 2: Thiol Imaging Cocktail for Alexa Fluor 647/CY5/CY3B/ATTO 488
  • Support Protocol 3: TCEP Imaging Cocktail for Alexa 750/Cy7
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunostaining of Cultured Cells

  Materials
  • BS‐C‐1 cells (African green monkey kidney, ATCC# CCL‐26)
  • Eagle's minimum essential medium (EMEM, ATCC, cat. no. 30‐2003)
  • Extraction solution (see recipe)
  • PFA/GA fixation solution (when using antibodies against tubulin or PMP70) or PFA‐only fixation solution (for clathrin) (see reciperecipes)
  • Phosphate buffered saline (PBS), pH 7.4 (Invitrogen, cat. no. 10010‐023)
  • 1 mg/ml (26 mM) sodium borohydride (NaBH 4) solution in water, prepared immediately prior to use
  • Storage solution (see recipe), optional
  • Block/Perm buffer (see recipe)
  • Primary antibody solution in Block/Perm buffer containing one of the following:
    • For microtubules (two‐color): rat anti‐alpha tubulin YL1/2 (Pierce, cat. no. MA1‐80017, 1 mg/ml) diluted 1:100 to 10 μg/ml and rabbit anti‐detyrosinated tubulin (Abcam, cat. no. Ab48389, whole serum) diluted 1:500
    • For peroxisomes: rabbit anti‐PMP70 (Invitrogen, cat. no. 78‐3100, 500 μg/ml) diluted 1:500 to 1 μg/ml
    • For clathrin: mouse anti‐clathrin X‐22 (Abcam, cat. no. ab2731, 6 mg/ml) diluted 1:1500 to 4 μg/ml
  • Secondary antibody solution (see protocol 2) in Block/Perm buffer containing one of the following:
    • For microtubules (two‐color): donkey anti‐rat (Jackson, cat. no. 712‐005‐150) Alexa Fluor 750 conjugate diluted to 2 μg/ml and donkey anti‐rabbit (Jackson, cat. no. 711‐005‐152) Alexa Fluor 647 conjugate diluted to 2 μg/ml
    • For peroxisomes: donkey anti‐rabbit (Jackson, cat. no. 711‐005‐152) Alexa Fluor 647 conjugate diluted to 2 μg/ml
    • For clathrin: donkey anti‐mouse (Jackson, cat. no. 715‐005‐151) Alexa Fluor 647 conjugate diluted to 2 μg/ml
  • Postfixation solution (see recipe)
  • 8‐well, no. 1.5 coverglass chamber (Ibidi μ‐Slide, cat. no. 80821 or Nunc Labtek II, cat. no. 155409)
  • 37ºC, 5% CO 2 humidified incubator
  • Humidity chamber at 25ºC (petri dish containing wet cloth)
  • Fluorescence microscope

Support Protocol 1: Secondary Antibody Labeling with NHS Fluorophores

  Materials
  • Anhydrous dimethyl sulfoxide (DMSO)
  • Alexa Fluor 750, NHS ester (Thermo Fisher/Life Technologies, cat. no. A‐20111)
  • Alexa Fluor 647, NHS ester (Thermo Fisher/Life Technologies, cat. no. A‐20006)
  • Cy3B, NHS ester (GE Healthcare, cat. no. PA63101)
  • ATTO 488, NHS ester (Atto‐Tec, cat. no. AD 488‐31)
  • Donkey anti‐rat (min Mouse), unconjugated secondary antibody (Jackson, cat. no. 712‐005‐153), or donkey anti‐rabbit, unconjugated secondary antibody (Jackson, cat. no. 711‐005‐152), or donkey anti‐mouse (min Rat), unconjugated secondary antibody (Jackson, cat. no. 715‐005‐151)
  • 1 M sodium bicarbonate (NaHCO 3) stock solution, freshly prepared
  • Phosphate buffered saline (PBS), pH 7.4 (Invitrogen, cat. no. 10010‐023)
  • Illustra NAP‐5 columns, Sephadex G‐25 (GE Healthcare, cat. no. 17‐0853‐02)
  • UV‐vis spectrometer and reduced volume cuvette

Support Protocol 2: Thiol Imaging Cocktail for Alexa Fluor 647/CY5/CY3B/ATTO 488

  Materials
  • Glucose oxidase enzyme powder from Aspergillus Niger (Sigma, cat. no. G2133)
  • Catalase from bovine liver (Sigma, cat. no. C100)
  • Phosphate buffered saline (PBS), pH 7.4 (Invitrogen, cat. no. 10010‐023)
  • 50% (w/v) glucose solution
  • 1 M Tris·Cl buffer, pH 8
  • Beta‐mercaptoethanol (BME, Sigma, cat. no. M6250)
  • Storage solution (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge

Support Protocol 3: TCEP Imaging Cocktail for Alexa 750/Cy7

  Materials
  • 1 M Tris·Cl buffer, pH 9
  • 50% (w/v) glucose solution
  • 100 mM methyl viologen (Sigma, cat. no. 856177), freshly prepared
  • 100 mM ascorbic acid (Fisher, cat. no. A61), freshly prepared
  • GLOX oxygen scavenger stock solution (see protocol 3)
  • 0.5 M TCEP, pH 7 ampule (Sigma, cat. no. 646547), use within 24 hr
  • Widefield fluorescence microscope
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Figures

Videos

Literature Cited

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