Skip to main content
SpringerLink
Log in

Immunocytochemical Fluorescent In Situ Visualization of Proteins In Arabidopsis

  • Protocol
  • First Online:
Arabidopsis Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1062))

Abstract

The understanding of cellular and subcellular functions often relies on the ability to visualize proteins as close as possible to their endogenous locations. A number of immunocytochemical techniques have been developed to detect proteins in situ using specific antibodies raised against proteins of interest. Here, we describe in detail two protocols commonly, successfully employed in Arabidopsis research. The first allows for immunolocalization of proteins in whole-mount Arabidopsis roots without the need for physical sectioning. The second allows for immunolocalization of proteins on semi-thin microtome sections of wax-embedded swamples. This approach is particularly useful when sectioning of Arabidopsis roots or other thicker plant organs is required for immunolocalization. We provide step-by-step protocols with extensive troubleshooting for both the whole-mount and sectioning protocols. Furthermore, critical steps, advantages, and limitations of the two protocols described here are discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94:2122–2127

    Article  PubMed  CAS  Google Scholar 

  2. Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci USA 97:3718–3723

    Article  PubMed  CAS  Google Scholar 

  3. Brandizzi F, Fricker M, Hawes C (2002) A greener world: the revolution in plant bioimaging. Nat Rev Mol Cell Biol 3:520–530

    Article  PubMed  CAS  Google Scholar 

  4. Held MA, Boulaflous A, Brandizzi F (2008) Advances in fluorescent protein-based imaging for the analysis of plant endomembranes. Plant Physiol 147:1469–1481

    Article  PubMed  CAS  Google Scholar 

  5. Iyer-Pascuzzi AS, Benfey PN (2010) Fluorescence-activated cell sorting in plant developmental biology. Methods Mol Biol 655:313–319

    Article  PubMed  CAS  Google Scholar 

  6. Ckurshumova W, Caragea AE, Goldstein RS, Berleth T (2011) Glow in the dark: fluorescent proteins as cell and tissue-specific markers in plants. Mol Plant 4:794–804

    Article  PubMed  CAS  Google Scholar 

  7. Webb MC, Gunning BES (1990) Embryo sac development in Arabidopsis thaliana L. megasporogenesis, including the microtubular cytoskeleton. Sex Plant Reprod 3:244–256

    Article  Google Scholar 

  8. Webb MC, Gunning BES (1991) The microtubular cytoskeleton during development of the zygote, proembryo and free-nuclear endosperm in Arabidopsis thaliana (L.) Heynh. Planta 184:187–195

    Article  Google Scholar 

  9. Hauser MT, Morikami A, Benfey PN (1995) Conditional root expansion mutants of Arabidopsis. Development 121:1237–1252

    PubMed  CAS  Google Scholar 

  10. Lauber MH, Waizenegger I, Steinmann T, Schwarz H, Mayer U, Hwang I, Lukowitz W, Jürgens G (1997) The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J Cell Biol 139:1485–1493

    Article  PubMed  CAS  Google Scholar 

  11. Steinmann T, Geldner N, Grebe M, Mangold S, Jackson CL, Paris S, Gälweiler L, Palme K, Jürgens G (1999) Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF. Science 286:316–318

    Article  PubMed  CAS  Google Scholar 

  12. Waizenegger I, Lukowitz W, Assaad F, Schwarz H, Jürgens G, Mayer U (2000) The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis. Curr Biol 10:1371–1374

    Article  PubMed  CAS  Google Scholar 

  13. Grebe M, Xu J, Möbius W, Ueda T, Nakano A, Geuze HJ, Rook MB, Scheres B (2003) Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes. Curr Biol 13:1378–1387

    Article  PubMed  CAS  Google Scholar 

  14. Friml J, Benková E, Mayer U, Palme K, Muster G (2003) Automated whole mount localisation techniques for plant seedlings. Plant J 34:115–124

    Article  PubMed  CAS  Google Scholar 

  15. Paciorek T, Sauer M, Balla J, Wiśniewska J, Friml J (2006) Immunocytochemical technique for protein localization in sections of plant tissues. Nat Protoc 1:104–107

    Article  PubMed  CAS  Google Scholar 

  16. Sauer M, Paciorek T, Benková E, Friml J (2006) Immunocytochemical techniques for whole-mount in situ protein localization in plants. Nat Protoc 1:98–103

    Article  PubMed  CAS  Google Scholar 

  17. Sauer M, Friml J (2010) Immunolocalization of proteins in plants. Methods Mol Biol 655:253–263

    Article  PubMed  CAS  Google Scholar 

  18. Dong Z, McCully ME, Canny MJ (1994) Retention of vacuole contents of plant cells during fixation. J Microsc 175:222–228

    Article  CAS  Google Scholar 

  19. Boenisch T (1999) Diluent buffer ions and pH: their influence on the performance of monoclonal antibodies in immunohistochemistry. Appl Immunohistochem Mol Morphol 7:300–306

    Article  CAS  Google Scholar 

  20. Kiss JZ, Giddings TH Jr, Staehelin LA, Sack FD (1990) Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis. Protoplasma 157:64–74

    Article  PubMed  CAS  Google Scholar 

  21. Lería F, Marco R, Medina FJ (2004) Structural and antigenic preservation of plant samples by microwave-enhanced fixation, using dedicated hardware, minimizing heat-related effects. Microsc Res Tech 65:86–100

    Article  PubMed  Google Scholar 

  22. Kuo J (2007) Processing plant tissues for ultrastructural study. Methods Mol Biol 369:35–45

    Article  PubMed  CAS  Google Scholar 

  23. Ripper D, Schwarz H, Stierhof YD (2008) Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration. Biol Cell 100:109–123

    Article  PubMed  CAS  Google Scholar 

  24. Stierhof YD, El Kasmi F (2010) Strategies to improve the antigenicity, ultrastructure preservation and visibility of trafficking compartments in Arabidopsis tissue. Eur J Cell Biol 89:285–297

    Article  PubMed  CAS  Google Scholar 

  25. Geldner N, Friml J, Stierhof YD, Jürgens G, Palme K (2001) Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 413:425–428

    Article  PubMed  CAS  Google Scholar 

  26. Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, Ueda T, Nakano A, Jürgens G (2003) The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112:219–230

    Article  PubMed  CAS  Google Scholar 

  27. Swarup R, Friml J, Marchant A, Ljung K, Sandberg G, Palme K, Bennett M (2001) Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Genes Dev 15:2648–2653

    Article  PubMed  CAS  Google Scholar 

  28. Willemsen V, Friml J, Grebe M, van den Toorn A, Palme K, Scheres B (2003) Cell polarity and PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function. Plant Cell 15:612–625

    Article  PubMed  CAS  Google Scholar 

  29. Men S, Boutté Y, Ikeda Y, Li X, Palme K, Stierhof YD, Hartmann MA, Moritz T, Grebe M (2008) Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity. Nat Cell Biol 10:237–244

    Article  PubMed  CAS  Google Scholar 

  30. Boutté Y, Frescatada-Rosa M, Men S, Chow CM, Ebine K, Gustavsson A, Johansson L, Ueda T, Moore I, Jürgens G, Grebe M (2010) Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis. EMBO J 29:546–558

    Article  PubMed  Google Scholar 

  31. Boutté Y, Men S, Grebe M (2011) Fluorescent in situ visualization of sterols in Arabidopsis roots. Nat Protoc 6:446–456

    Article  PubMed  Google Scholar 

  32. Fischer U, Ikeda Y, Ljung K, Serralbo O, Singh M, Heidstra R, Palme K, Scheres B, Grebe M (2006) Vectorial information for Arabidopsis planar polarity is mediated by combined AUX1, EIN2, and GNOM activity. Curr Biol 16:2143–2149

    Article  PubMed  CAS  Google Scholar 

  33. Singh SK, Fischer U, Singh M, Grebe M, Marchant A (2008) Insight into the early steps of root hair formation revealed by the procuste1 cellulose synthase mutant of Arabidopsis thaliana. BMC Plant Biol 8:57

    Article  PubMed  Google Scholar 

  34. Ikeda Y, Men S, Fischer U, Stepanova AN, Alonso JM, Ljung K, Grebe M (2009) Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis. Nat Cell Biol 11:31–38

    Article  Google Scholar 

  35. Boutté Y, Crosnier MT, Carraro N, Traas J, Satiat-Jeunemaitre B (2006) The plasma membrane recycling pathway and cell polarity in plants: studies on PIN proteins. J Cell Sci 119:1255–1265

    Article  PubMed  Google Scholar 

  36. Brown RC, Lemmon BE, Mullinax JB (1989) Immunofluorescent staining of microtubules in plant tissues: improved embedding and sectioning techniques using polyethylene glycol (PEG) and Steedman’s wax. Bot Acta 102:54–61

    Google Scholar 

  37. Vitha S, Baluska F, Mews M, Volkmann D (1997) Immunofluorescence detection of F-actin on low melting point wax sections from plant tissues. J Histochem Cytochem 45:89–95

    Article  PubMed  CAS  Google Scholar 

  38. Carraro N, Forestan C, Canova S, Traas J, Varotto S (2006) ZmPIN1a and ZmPIN1b encode two novel putative candidates for polar auxin transport and plant architecture determination of maize. Plant Physiol 142:254–264

    Article  PubMed  CAS  Google Scholar 

  39. de Reuille PB, Bohn-Courseau I, Ljung K, Morin H, Carraro N, Godin C, Traas J (2006) Computer simulations reveal properties of the cell-cell signaling network at the shoot apex in Arabidopsis. Proc Natl Acad Sci USA 103:1627–1632

    Article  PubMed  Google Scholar 

  40. Gendre D, Oh J, Boutté Y, Best JG, Samuels L, Nilsson R, Uemura T, Marchant A, Bennett MJ, Grebe M, Bhalerao RP (2011) Conserved Arabidopsis ECHIDNA protein mediates trans-Golgi-network trafficking and cell elongation. Proc Natl Acad Sci USA 108:8048–8053

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank David Ehrhardt (Stanford, USA) for making available EGFP-LTI6a seeds, Gerd Jürgens (Tübingen, Germany) for providing anti-KNOLLE serum, and Ian Moore (Oxford, UK) for N-ST-YFP.

Author information

Authors and Affiliations

  1. Department of Forest Genetics and Plant Physiology, UPSC, Swedish University of Agricultural Sciences, Umeå, Sweden

    Yohann Boutté

  2. Membrane biogenesis laboratory, CNRS, UMR5200, Victor Ségalen Bordeaux 2 University, Bordeaux, France

    Yohann Boutté

  3. Department of Plant Physiology, Umeå Plant Science Centre (UPSC), Umeå University, Fysiologihuset Byggnad L, Umeå, Sweden

    Markus Grebe

Authors
  1. Yohann Boutté
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Markus Grebe
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. CSIC Centro Nacional de Biotecnología, Madrid, Spain

    Jose J. Sanchez-Serrano

  2. Depto. Biologia de Plantas, CSIC Madrid Centro de Investigaciones Biologicas, Madrid, Spain

    Julio Salinas

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Boutté, Y., Grebe, M. (2014). Immunocytochemical Fluorescent In Situ Visualization of Proteins In Arabidopsis . In: Sanchez-Serrano, J., Salinas, J. (eds) Arabidopsis Protocols. Methods in Molecular Biology, vol 1062. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-580-4_24

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-580-4_24

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-579-8

  • Online ISBN: 978-1-62703-580-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation