Visualizing G Protein‐Coupled Receptor‐Receptor Interactions in Brain Using Proximity Ligation In Situ Assay

Jaume Taura1, Víctor Fernández‐Dueñas1, Francisco Ciruela2

1 Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL‐Universitat de Barcelona, Barcelona, 2 Department of Physiology, Faculty of Sciences, University of Ghent, Ghent
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.17
DOI:  10.1002/0471143030.cb1717s67
Online Posting Date:  June, 2015
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Abstract

G protein‐coupled receptors (GPCRs) constitute the largest family of plasma membrane receptors, thus representing the more investigated drug targets in the design of new therapeutic strategies. The existence of receptor‐receptor interactions has revolutionized the field, since GPCR oligomerization might confer new intervention opportunities in pharmacotherapy. However, demonstrating the existence of such receptor‐receptor interactions in native tissue has been a bottleneck in GPCR pharmacology. Here, we discuss an experimental approach, the proximity ligation in situ assay (P‐LISA), which provides enough sensitivity to evaluate a receptor's close proximity within a named GPCR oligomer. Indeed, we provide a detailed step‐by‐step protocol for P‐LISA experiments visualizing receptor‐receptor interactions in brain slices. Additionally, we provide instructions for slide observation, data acquisition and quantification. Finally, we also discuss these critical aspects determining the success of the technique, namely the fixation process and the validation of the primary antibodies used. Overall, the P‐LISA is a powerful and straightforward technique to visualize receptor‐receptor interactions when performed under optimal conditions. © 2015 by John Wiley & Sons, Inc.

Keywords: GPCR; proximity ligation assay; brain; oligomerization

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Visualization of GPCR Oligomers by Proximity Ligation In Situ Assay
  • Support Protocol 1: Slide Observation, Image Acquisition, and Quantifcation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Visualization of GPCR Oligomers by Proximity Ligation In Situ Assay

  Materials
  • CD1 mouse (in‐house colony)
  • Ketamine (50 mg/ml; Ketalar, Panpharma)
  • Xylazine (20 mg/ml; Rompun, Bayer)
  • 70% ethanol
  • 4% formaldehyde solution (see recipe)
  • 1× PBS (see recipe)
  • Walter's antifreezing solution (see recipe)
  • Blocking solution:
    • 10% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories)
    • and 0.05% Triton X‐100 (Sigma‐Aldrich) in 1× PBS
  • Normal donkey serum (NDS; Jackson ImmunoResearch Laboratories)
  • Duolink in situ PLA probe anti‐goat PLUS (Sigma‐Aldrich)
  • Duolink in situ PLA probe anti‐rabbit MINUS (Sigma‐Aldrich)
  • 1× ligation solution (see recipe)
  • T4 DNA ligase (1 U/μl; Promega)
  • P‐LISA wash buffer A (see recipe)
  • 1× rolling‐circle amplification (RCA) solution
  • phi29 DNA polymerase (Life Technologies)
  • P‐LISA wash buffer B (see recipe)
  • Duolink in situ mounting medium containing 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich)
  • Transparent nail polish
  • Surgical scissors
  • Forceps
  • 27‐gauge needle
  • Rack (for dissection)
  • Tape or pins
  • 1‐cc syringe
  • 25‐gauge, 25‐mm perfusion needle
  • Vibratome (VT1200, Leica Lasertechnik GmbH)
  • Round brush, sizes 10/0 to 2/10
  • Microscope slides and coverslips

Support Protocol 1: Slide Observation, Image Acquisition, and Quantifcation

  Materials
  • Laser scanning confocal microscope (LSCM; TCS 4D, Leica Lasertechnik GmbH)
  • Fiji program (Fiji Is Just ImageJ; http://fiji.sc/Fiji)
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Figures

Videos

Literature Cited

Literature Cited
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