In Situ Proximity Ligation Assay for Microscopy and Flow Cytometry

Karl‐Johan Leuchowius1, Irene Weibrecht1, Ola Söderberg1

1 Department of Immunology, Genetics, and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.36
DOI:  10.1002/0471142956.cy0936s56
Online Posting Date:  April, 2011
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Abstract

The ability to study proteins and protein interactions inside cells and tissues is important for elucidating how cells function in health and disease. The in situ proximity ligation assay (in situ PLA) presented here can be used to visualize proteins, protein‐protein interactions, and post‐translational modifications in cells and tissues. The method is based upon the use of antibodies that target the proteins involved in an interaction; hence, the method has the advantage that it can be used in clinical specimens, providing localized, quantifiable single molecule detection in single cells. This unit describes how in situ PLA can be used with fluorescence microscopy and flow cytometry to study proteins (obtaining high sensitivity and specificity of detection) and protein interactions. It also includes information on expected results and information on how to troubleshoot the assay. Curr. Protoc. Cytom. 56:9.36.1‐9.36.15. © 2011 by John Wiley & Sons, Inc.

Keywords: in situ; proximity ligation; protein interactions; post‐translational modifications

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Situ PLA for Microscopy
  • Basic Protocol 2: In Situ PLA for Flow Cytometry
  • Support Protocol 1: Conjugation of Oligonucleotides to Antibodies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Situ PLA for Microscopy

  Materials
  • Cultured cells
  • Phosphate‐buffered saline (PBS; see recipe)
  • Fixative (e.g., ethanol or paraformaldehyde)
  • Tris‐buffered saline (TBS), pH 7.4
  • 0.5% Tween‐20, diluted in water
  • Blocking solution (see recipe)
  • Proximity probe incubation buffer (see recipe)
  • Proximity probe type 1 (see protocol 3 for details)
  • Proximity probe type 2 (see protocol 3 for details)
  • Wash buffer (see recipe)
  • Ligation mixture (see recipe)
  • T4 DNA ligase (1 U/µl; Fermentas)
  • Rolling circle amplification mixture (see recipe)
  • Phi‐29 polymerase (Fermentas)
  • Detection mixture (see recipe)
  • Mounting medium (e.g., VectaShield, Vector Labs)
  • Microscopy glass slides (e.g., 8‐well slides)
  • Incubator (preferably at +37°C)
  • Pen or mask for delineation of reaction areas (e.g., grease pen or silicon mask)
  • Humidity chamber, e.g., a small box with wet paper towels in the bottom
  • Vortex
  • Washing/staining jars
  • Fluorescence microscope with excitation/emission filters compatible with fluorophore and nuclear stain excitation/emission spectra
  • Image analysis software (e.g., BlobFinder or Cellprofiler)

Basic Protocol 2: In Situ PLA for Flow Cytometry

  Materials
  • Cells of interest
  • Trypsin or Accutase
  • Fixative (e.g., ethanol or paraformaldehyde)
  • Proximity probe incubation buffer (see recipe)
  • Proximity probe type 1 (see protocol 3 for details)
  • Proximity probe type 2 (see protocol 3 for details)
  • Wash buffer (see recipe)
  • Ligation mixture (see recipe)
  • T4 DNA ligase (1 U/µl; Fermentas)
  • Rolling circle amplification mixture (see recipe)
  • Phi‐29 polymerase (Fermentas)
  • Detection buffer (see recipe)
  • FACS‐tubes or V‐bottomed 96‐well plates
  • Vortex
  • Centrifuge
  • Orbital shaker
  • Flow cytometer, with lasers and filters appropriate for the fluorophore used for the detection oligonucleotide

Support Protocol 1: Conjugation of Oligonucleotides to Antibodies

  Materials
  • Antibody of interest (2.0 mg/ml or higher) in an amine‐free buffer (e.g., PBS, pH 7.4), without carrier proteins
  • Succinimidyl 4‐hydrazinonicotinate acetone hydrazone (SANH)
  • Dimethyl sulfoxide (DMSO)
  • Conjugation buffer (see recipe)
  • Aldehyde‐modified oligonucleotide:
    • Type 1 oligonucleotide: 5′‐aldehyde‐AAA AAA AAA ATA TGA CAG AAC TAG ACA CTC TT
    • Type 2 oligonucleotide: 5′‐aldehyde‐AAA AAA AAA AGA CGC TAA TAG TTA AGA CGC TT)
  • Aniline
  • Desalting device (e.g., Zeba Desalt Spin Columns by Thermo Scientific, or illustra MicroSpin G‐50 by GE Healthcare)
  • HPLC with a Superdex‐75 or Superdex‐100 column
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Figures

Videos

Literature Cited

Literature Cited
   Allalou, A. and Wählby, C. 2009. BlobFinder, a tool for fluorescence microscopy image cytometry. Comput. Methods Programs Biomed. 94:58‐65.
   Baan, B., Pardali, E., ten Dijke, P., and van Dam, H. 2010. In situ proximity ligation detection of c‐Jun/AP‐1 dimers reveals increased levels of c‐Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo. Mol. Cell Proteomics 9:1982‐1990.
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   Frolova, E.I., Gorchakov, R., Pereboeva, L., Atasheva, S., and Frolov, I. 2010. Functional Sindbis virus replicative complexes are formed at the plasma membrane. J. Virol. 84:11679‐11695.
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   Johansson, H., Svensson, F., Runnberg, R., Simonsson, T., and Simonsson, S. 2010. Phosphorylated nucleolin interacts with translationally controlled tumor protein during mitosis and with Oct4 during interphase in ES cells. PLoS One 5:e13678.
   Kenworthy, A.K. 2001. Imaging protein‐protein interactions using fluorescence resonance energy transfer microscopy. Methods 24:289‐296.
   Lasserre, R., Charrin, S., Cuche, C., Danckaert, A., Thoulouze, M.I., de Chaumont, F., Duong, T., Perrault, N., Varin‐Blank, N., Olivo‐Marin, J.C., Etienne‐Manneville, S., Arpin, M., Di Bartolo, V., and Alcover, A. 2010. Ezrin tunes T‐cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse. EMBO J. 29:2301‐2314.
   Leuchowius, K.J., Weibrecht, I., Landegren, U., Gedda, L., and Söderberg, O. 2009. Flow cytometric in situ proximity ligation analyses of protein interactions and post‐translational modification of the epidermal growth factor receptor family. Cytometry A 75:833‐839.
   Leuchowius, K.J., Jarvius, M., Wickström, M., Rickardson, L., Landegren, U., Larsson, R., Söderberg, O., Fryknas, M., and Jarvius, J. 2010. High content screening for inhibitors of protein interactions and post‐translational modifications in primary cells by proximity ligation. Mol. Cell Proteomics 9:178‐183.
   Sehat, B., Tofigh, A., Lin, Y., Trocme, E., Liljedahl, U., Lagergren, J., and Larsson, O. 2010. SUMOylation mediates the nuclear translocation and signaling of the IGF‐1 receptor. Sci. Signal 3:ra10.
   Söderberg, O., Gullberg, M., Jarvius, M., Ridderstråle, K., Leuchowius, K.J., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L.G., and Landegren, U. 2006. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3:995‐1000.
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