Detection of Receptor Heteromerization Using In Situ Proximity Ligation Assay

Ivone Gomes1, Salvador Sierra1, Lakshmi A. Devi1

1 Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.16
DOI:  10.1002/cpph.15
Online Posting Date:  December, 2016
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Abstract

Although G protein‐coupled receptor (GPCR) heteromerization has been extensively demonstrated in vitro using heterologous cells that overexpress epitope‐tagged receptors, their presence in endogenous systems is less well established. This is because a criterion to identify receptor heteromerization is the demonstration that the two interacting receptors are present not only in the same cell but also in the same subcellular compartment in close enough proximity to allow for direct receptor‐receptor interaction. This has been difficult to study in native tissues due to a lack of sensitive and selective tools not only capable of detecting low‐abundance proteins but also of demonstrating that they are in sufficiently close proximity to interact. The latter can be achieved using a proximity ligation assay (PLA). Detailed in this unit are protocols for demonstrating the presence of GPCR heteromers in endogenous cells as well as animal and human tissues, the controls required for these assays, and troubleshooting tips. © 2016 by John Wiley & Sons, Inc.

Keywords: dimerization; G‐protein coupled receptors; in situ proximity ligation assay; receptor heteromers

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Indirect PLA in Mouse Tissue Slices
  • Basic Protocol 2: Indirect PLA for Cells
  • Basic Protocol 3: Indirect PLA Using Formalin‐Fixed Paraffin‐Embedded Human Brain Sections
  • Basic Protocol 4: Direct PLA for Tissue Slices or Cells
  • Support Protocol 1: Preparation of Tissue Sections for PLA
  • Support Protocol 2: Preparation of Cells to be Used for PLA
  • Support Protocol 3: Conjugation of Primary Antibodies to Oligonucleotide Probes Plus or Minus
  • Support Protocol 4: Image Acquisition and Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Indirect PLA in Mouse Tissue Slices

  Materials
  • Fixed free‐floating mouse brain tissue slices co‐expressing both of the receptor protomers being investigated or expressing only individual protomers ( protocol 5)
  • 1× phosphate‐buffered saline (1× PBS; see recipe for 10×)
  • Gelatin from bovine skin (Sigma‐Aldrich, cat. no. G9391)
  • 0.05 M Tris⋅Cl, pH 7.6 ( appendix 2A)
  • Triton X‐100 (Sigma‐Aldrich, cat. no. X‐100)
  • PLA probes (species‐specific secondary antibodies conjugated to either PLUS or MINUS oligonucleotide sequences): every PLA probe kit includes the blocking and antibody diluent solutions (Sigma‐Aldrich, cat. no. DUO92001 to DUO 92010; check company Web site) in addition to the conjugated secondary antibodies (the method for determining the selectivity of the PLA probes is described in Critical Parameters)
  • Primary antibodies that selectively recognize each GPCR protomer of interest: these may be either obtained commercially or generated and validated in‐house (the technique for determining the selectivity of antibodies for a protomer is described below in Critical Parameters)
  • Duolink In Situ Wash Buffer (Fluorescence) comprising PLA Buffer A and Buffer B (Sigma‐Aldrich, cat. no. DUO82049), or equivalent reagents prepared in‐house (see reciperecipes)
  • Duolink In Situ Detection Reagent Red (includes ligase, ligase reagent, polymerase, and amplification reagent; Sigma‐ Aldrich, cat. no. DUO92008; for fluorophores different from Red, check the Web site)
  • 0.01× PLA Buffer B (see recipe)
  • Duolink In Situ Mounting Medium with DAPI (Sigma‐Aldrich, cat. no. DUO82040)
  • Nail polish or acrylic resin
  • Superfrost Plus Microscope Slides (Fisher Scientific, cat. no. 22‐037‐246)
  • Hydrophobic pen (Vector Laboratories, cat. no. H‐4000)
  • Coplin jars (Sigma‐Aldrich, cat. no. S5766‐6EA)
  • Orbital shaker
  • Opaque Humidity Chamber Plus (Biolegend, cat. no. 926301)
  • Blotting paper
  • Tweezers
  • Coverslips (glass)
  • Leica TCS SP5 confocal laser scanning microscope (405‐nm laser line for DAPI, 568‐nm laser line for Detection Reagent Red) 

Basic Protocol 2: Indirect PLA for Cells

  Materials
  • Cells endogenously expressing one or both receptor protomers being investigated, plated and processed as in protocol 6
  • PLA probes (species‐specific secondary antibodies conjugated to either PLUS or MINUS oligonucleotide sequences): every PLA probe kit includes the blocking and antibody diluent solutions (Sigma‐Aldrich, cat. no. DUO92001 to DUO 92010; check company Web site) in addition to the conjugated secondary antibodies (the method for determining the selectivity of the PLA probes is described in Critical Parameters)
  • Primary antibodies from a commercial supplier or generated and validated in‐house, that selectively recognize each protomer being investigated (described in Critical Parameters is a procedure for determining the selectivity of the antibodies for a target protomer)
  • Duolink In Situ Wash Buffer (Fluorescence) comprising PLA Buffer A and Buffer B (Sigma‐Aldrich, cat. no. DUO82049), or equivalent reagents prepared in‐house (see reciperecipes)
  • 0.01× PLA Buffer B (see recipe)
  • Duolink In Situ Detection Reagent Red (includes ligase, ligation reagent, polymerase, and amplification reagent; Sigma‐ Aldrich, cat. no. DUO92008; for fluorophores different from Red, check the Web site)
  • Duolink In Situ Mounting medium with DAPI (Sigma‐Aldrich, cat. no. DUO82040)
  • Nail polish or acrylic resin
  • Blotting paper
  • Coverslips (glass)
  • Leica TCS SP5 confocal laser scanning microscope (405 nm laser line for DAPI, 568 nm laser line for Detection Reagent Red)
  • Additional reagents and equipment for plating and processing cells for PLA ( protocol 6)

Basic Protocol 3: Indirect PLA Using Formalin‐Fixed Paraffin‐Embedded Human Brain Sections

  Materials
  • Formalin fixed‐paraffin embedded human brain sections (these were a kind gift from from Dr. Marco Hefti, Department of Pathology, Mount Sinai Hospital, New York)
  • Xylene (mixed isomers; Fisher Scientific, cat. no. X5‐1)
  • 100%, 95%, 75%, and 50% ethanol
  • 10 mM sodium citrate solution containing 0.05% (v/v) Tween 20
  • 0.1% Sudan Black B in 70% ethanol solution (see recipe)
  • Duolink In Situ Wash Buffer (Fluorescence) comprising PLA Buffer A and Buffer B (Sigma‐Aldrich, cat. no. DUO82049), or equivalent reagents prepared in‐house (see reciperecipes)
  • 0.01× PLA Buffer B
  • Duolink In Situ Mounting Medium with DAPI (Sigma‐Aldrich, cat. no. DUO82040)
  • Hydrophobic pen (Vector Laboratories, cat. no. H‐4000)
  • 10‐ml ml syringe
  • Tweezers
  • Coverslips (glass)
  • Leica TCS SP5 confocal laser scanning microscope (405 nm laser line for DAPI, 568 nm laser line for Detection Reagent Red)
  • Additional reagents and equipment for indirect PLA (see protocol 1, steps 6 to 19)

Basic Protocol 4: Direct PLA for Tissue Slices or Cells

  Materials
  • Fixed free‐floating mouse brain tissue slices/cells co‐expressing both receptor protomers of interest or expressing only individual protomers
  • Primary antibodies coupled to oligonucleotide sequence PLUS or MINUS (see protocol 7)
  • Duolink In Situ Wash Buffer (Fluorescence) comprising PLA Buffer A and Buffer B (Sigma‐Aldrich, cat. no. DUO82049), or equivalent reagents prepared in‐house (see reciperecipes)
  • 0.01× PLA Buffer B
  • Duolink In Situ Detection Reagent Red (includes ligase, ligation reagent, polymerase, and amplification reagent; Sigma‐ Aldrich, cat. no. DUO92008; for fluorophores different from Red, check the Web site)
  • Duolink In Situ Mounting Medium with DAPI (Sigma‐Aldrich, cat no. DUO82040)
  • Nail polish or acrylic resin
  • Leica TCS SP5 confocal laser scanning microscope (405 nm laser line for DAPI, 568 nm laser line for Detection Reagent Red)
  • Additional reagents and equipment for indirect PLA assays ( protocol 1 for tissue slices or protocol 2 for cells)

Support Protocol 1: Preparation of Tissue Sections for PLA

  Materials
  • Mice (wild‐type, and lacking one of the receptor protomers; of the same strain background)
  • Ketamine hydrochloride/xylazine hydrochloride (Sigma‐Aldrich, cat. no. K113)
  • 1× Ringer's solution (see recipe)
  • 4% paraformaldehyde (see recipe)
  • 1× phosphate‐buffered saline (1× PBS; see recipe for 10×)
  • Cryoprotectant solution (see recipe)
  • Deltaphase isothermal pad (Braintree Scientific, Inc., cat. no. 39DP)
  • Surgical tape
  • Surgical work surface, e.g., Styrofoam sheet
  • Surgical instruments for mice
  • 18‐G needles
  • Peristaltic pump (Cole Parmer, cat. no. WU‐07525‐20)
  • Tubing for peristaltic pump
  • 22‐ to 23‐G blunt needles
  • Leica SM 2000 R sliding microtome with BFS‐3 MP Physitemp Temperature Controller (or dry ice tray) or Leica VT1000 S vibratome
  • Cryotubes
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Support Protocol 2: Preparation of Cells to be Used for PLA

  Materials
  • Cells endogenously expressing individual receptor protomers or co‐expressing the two protomers being investigated
  • Cell growth medium, e.g.:
  • F12 medium (Life Technologies, cat. no. 11765‐054) containing 10% FBS (BioTC, cat. no. FBS‐01) and penicillin‐streptomycin (Corning, cat. no. 30‐002‐CI) for CHO cells
  • DMEM (Corning Cellgro, cat. no. 10‐013‐CV) containing 10% FBS (BioTC, cat. no. FBS‐01) and penicillin‐streptomycin (Corning, cat. no.30‐002‐CI) for HEK‐293 cells
  • Poly‐D‐lysine (Sigma‐Aldrich; Cat. No. P6407)
  • 4% paraformaldehyde (see recipe)
  • 1× phosphate‐buffered saline (1× PBS; see recipe for 10×)
  • 1× PBS containing 20 mM glycine
  • 1× PBS containing 20 mM glycine and 0.05% Triton‐X‐100
  • Sterile 16‐well Lab‐Tek chamber slide with cover glass slide (Thermo Scientific, cat. no. 178599).
  • Leica TCS SP5 confocal laser scanning microscope (405 nm laser line for DAPI, 568 nm laser line for Detection Reagent Red)

Support Protocol 3: Conjugation of Primary Antibodies to Oligonucleotide Probes Plus or Minus

  Materials
  • Primary antibodies to each receptor protomer at a concentration of 1 mg/ml in 1× PBS.
  • Duolink In Situ Probemaker PLUS kit (Sigma‐Aldrich, cat. no. DUO92009): the box includes conjugation buffer (green lid), stop reagent (red lid), and storage solution (blue lid)
  • Duolink In Situ Probemaker MINUS kit (Sigma‐Aldrich, cat. no. DUO92010): the box includes conjugation buffer (green lid), stop reagent (red lid), and storage solution (blue lid)
  • 0.2‐ml PCR tubes (Thermo Scientific cat. no. AB0337)

Support Protocol 4: Image Acquisition and Data Analysis

  Materials
  • ImageJ, 64‐bit
  • Iterative Deconvolve 3‐D plugin (http://www.optinav.com/Iterative‐Deconvolve‐3D.htm)
  • Images from the above protocols
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Figures

Videos

Literature Cited

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