Abstract
Immunofluorescence microscopy reveals localization of proteins in cells and tissues by means of highly specific, fluorescently labeled antibodies. This technique is an important complement to localization methods that use genetically encoded fluorescent tags. This chapter describes the five stages of immunofluorescence localization of proteins in plant chloroplasts in sectioned leaf tissue: (1) fixation, (2) tissue embedding and sectioning, (3) treatment of sections prior to immunolabeling, (4) immunostaining, and (5) fluorescence microscopy and image capture. Protocols for both cryosectioning and sectioning of low-melting-point wax-embedded samples are described. Immunofluorescence localization in chloroplasts is complicated by their intense autofluorescence background. Measures to suppress nonspecific background staining, confirm specificity of the fluorescence signal, and optimize imaging conditions are described.
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Johnson, I. D. (2006) Practical considerations in the selection and application of fluorescent probes. In, Handbook of Biological Confocal Microscopy, 3rd edn. (Pawley, J., ed.) Springer, New York, USA, pp. 353–367.
Sauer, M., Paciorek, T., Benkova, E., and Friml, J. (2006) Immunocytochemical techniques for whole-mount in situ protein localization in plants. Nat. Protocols 1, 98–103.
McAndrew, R. S., Olson, B. J. S. C., Kadirjan-Kalbach, D. K., Chi-Ham, C. L., Vitha, S., Froehlich, J. E., and Osteryoung, K. W. (2008) In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex. Biochem. J. 412, 367–378.
Vitha, S., McAndrew, R. S., and Osteryoung, K. W. (2001) FtsZ ring formation at the chloroplast division site in plants. J. Cell Biol. 153, 111–119.
Wick, S. M., and Duniec, J. (1986) Effects of various fixatives on the reactivity of plant cell tubulin and calmodulin in immunofluorescence microscopy. Protoplasma 133, 1–18.
Sompuram, S. R., Vani, K., Messana, E., and Bogen, S. A. (2004) A molecular mechanism of formalin fixation and antigen retrieval. Am. J. Clin. Pathol. 121, 190–199.
Ferris, A. M., Giberson, R. T., Sanders, M. A., and Day, J. R. (2009) Advanced laboratory techniques for sample processing and immunolabeling using microwave radiation. J. Neurosci. Meth. 182, 157–164.
Nakazono, M., Qiu, F., Borsuk, L. A., and Schnable, P. S. (2003) Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 15, 583–596.
Feltlová, M. (2000) Preparing plant tissue cryosections for light microscopy – a little improvement. Biol. Listy 65, 270–272.
Slot, J. W., and Geuze, H. J. (2007) Cryosectioning and immunolabeling. Nat. Protocols 2, 2480–2491.
Steedman, H. F. (1957) A new ribboning embedding medium for histology. Nature 179, 1345.
Vitha, S., Baluška, F., Mews, M., and Volkmann, D. (1997) Immunofluorescence detection of F-actin on low melting point wax sections from plant tissues. J. Histochem. Cytochem. 45, 89–95.
McCartney, L., Marcus, S. E., and Knox, J. P. (2005) Monoclonal antibodies to plant cell wall xylans and arabinoxylans. J. Histochem. Cytochem. 53, 543–546.
Samaj, J., Ovecka, M., Hlavacka, A., Lecourieux, F., Meskiene, I., Lichtscheidl, I., Lenart, P., Salaj, J., Volkmann, D., Bogre, L., Baluska, F., and Hirt, H. (2002) Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth. EMBO J. 21, 3296–3306.
Paciorek, T., Sauer, M., Balla, J., Wisniewska, J., and Friml, J. (2006) Immunocytochemical technique for protein localization in sections of plant tissues. Nat. Protocols 1, 104–107.
Otali, D., Stockard, C. R., Oelschlager, D. K., Wan, W., Manne, U., Watts, S. A., and Grizzle, W. E. (2009) Combined effects of formalin fixation and tissue processing on immunorecognition. Biotech. Histochem. 84, 223–247.
D’Amico, F., Skarmoutsou, E., and Stivala, F. (2009) State of the art in antigen retrieval for immunohistochemistry. J. Immunol. Methods 341, 1–18.
Gong, H. Q., Peng, Y. B., Zou, C., Wang, D. H., Xu, Z. H., and Bai, S. N. (2006) A simple treatment to significantly increase signal specificity in immunohistochemistry. Plant Mol. Biol. Rep. 24, 93–101.
Vitha, S., Baluška, F., Jasik, J., Volkmann, D., and Barlow, P. (2000) Steedman’s wax for F-actin visualization. In, Actin: a Dynamic Framework for Multiple Plant Cell Functions (Staiger, C. J., Baluška, F., Volkmann, D., and Barlow, P., eds.) Kluwer, Dordrecht, The Netherlands, pp. 619–636.
Finney, M. (1998) Nonradioactive methods for visualization of protein blots. In, Immunochem-ical Protocols (Pound, J. D., ed.) Humana Press, Totowa, NJ, USA, pp. 207–216.
Orcutt, K. M., Ren, S. S., and Gundersen, K. (2009) Detecting proteins in highly autofluorescent cells using quantum dot antibody conjugates. Sensors-Basel 9, 7540–7549.
Collins, T. Mounting Media and Antifade Reagents, Collation of information from the Confocal listserver archives and the Histonet archives as well as other web-resources. http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Mountants.pdf.
Martini, N., Bewersdorf, J., and Hell, S. W. (2002) A new high-aperture glycerol immersion objective lens and its application to 3D-fluorescence microscopy, J. Microsc. 206, 146–151.
Egner, A., and Hell, S. W. (2006) Aberrations in confocal and multi-photon fluorescence microscopy induced by refractive index mismatch. In, Handbook of Biological Confocal Microscopy, 3rd edn. (Pawley, J., ed.) Springer, New York, USA, pp. 404–413.
Staudt, T., Lang, M. C., Medda, R., Engelhardt, J., and Hell, S. W. (2007) 2,2’-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc. Res. Techn. 70, 1–9.
Pawley, J. (2006) Handbook of Biological Confocal Microscopy, 3rd edn. Springer, New York, USA.
Wayne, R. (2009) Light and Video Microscopy, 1st edn. Academic Press, New York, USA.
Mackenzie, J. M., Burke, M. G., Carvalho, T., and Eades, A. (2006) Ethics and digital imaging. Microsc. Today 14, 40–41.
Cromey, D. (2010) Avoiding twisted pixels: ethical guidelines for the appropriate use and manipulation of scientific digital images. Sci. Eng. Ethics doi:10.1007/s11948-010-9201-y.
Tirichine, L., Andrey, P., Biot, E., Maurin, Y., and Gaudin, V. (2009) 3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei. Plant Methods 5, 11.
Koiwai, H., Nakaminami, K., Seo, M., Mitsuhashi, W., Toyomasu, T., and Koshiba, T. (2004) Tissue-specific localization of an abscisic acid biosynthetic enzyme, AAO3, in Arabidopsis. Plant Physiol. 134, 1697–1707.
Moreno, N., Bougourd, S., Haseloff, J., and Feijó, J. A. (2006) Imaging plant cells. In, Handbook of Biological Confocal Microscopy, 3rd edn. (Pawley, J., ed.) Springer, New York, USA, pp. 769–787.
Murray, J. M., Appleton, P. L., Swedlow, J. R., and Waters, J. C. (2007) Evaluating performance in three-dimensional fluorescence microscopy. J. Microsc. 228, 390–405.
Cannell, M. B., McMorland, A., and Soeller, C. (2006) Image enhancement by deconvolution. In, Handbook of Biological Confocal Microscopy, 3rd ed. (Pawley, J., ed.) Springer, New York, USA, pp. 488–500.
Y. Sun, Davis, P., Kosmacek, E. A., Ianzini, F., and Mackey, M. A. (2009) An open-source deconvolution software package for 3-D quantitative fluorescence microscopy imaging. J. Microsc. 236, 180–193.
Biggs, D. S. (2010) 3D deconvolution microscopy. Curr. Protoc. Cytom. 52, 12.19.11-12.19.20.
Lichtman, J. W., and Conchello, J.-A. (2005) Fluorescence microscopy. Nat. Methods 2, 910–919.
Ono, M., Murakami, T., Kudo, A., Isshiki, M., Sawada, H., and Segawa, A. (2001) Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy. J. Histochem. Cytochem. 49, 305–312.
Vitha, S., Bryant, V. M., Zwa, A., and Holzenburg, A. (2010) 3D confocal imaging of pollen. Microsc. Today 18, 26–28.
Keller, H. E. (2006) Objective lenses for confocal microscopy. In, Handbook of Biological Confocal Microscopy, 3rd edn. (Pawley, J., ed.) Springer, New York, USA, pp. 145–161.
Stuurman, N., Amodaj N., and Vale, R. D. (2007) Micro-Manager: open source software for light microscope imaging, Microsc. Today 15, 42–43.
Vitha, S., Froehlich, J. E., Koksharova, O., Pyke, K. A., van Erp, H., and Osteryoung, K. W. (2003) ARC6 Is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2. Plant Cell 15, 1918–1933.
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Preparation of this manuscript was supported by grants from the National Science Foundation and US Dept. of Energy to K.W.O.
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Vitha, S., Osteryoung, K.W. (2011). Immunofluorescence Microscopy for Localization of Arabidopsis Chloroplast Proteins. In: Jarvis, R. (eds) Chloroplast Research in Arabidopsis. Methods in Molecular Biology, vol 774. Humana Press. https://doi.org/10.1007/978-1-61779-234-2_3
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DOI: https://doi.org/10.1007/978-1-61779-234-2_3
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