Immunocytochemistry of plant cells

Immunocytochemistry of Plant Cells Kevin Vaughn Immunocytochemistry of Plant Cells 123 Kevin Vaughn Salem, OR USA ISBN 978-94-007-6060-8 DOI 10.1007/978-94-007-6061-5 ISBN 978-94-007-6061-5 (eBook) Springer Dordrecht Heidelberg New York London Library of Congress Control Number: 2012956166 Ó Springer Science?Business Media Dordrecht 2013 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer 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express or implied, with respect to the material contained herein Printed on acid-free paper Springer is part of Springer Science?Business Media (www.springer.com) Preface Immunocytochemistry of plant cells has come a long way from the first review on this subject by Bruce Knox in the early 1980s In that early review, our only tools were fluorescein-labeled antibodies for light microscopy and ferritin-labeled antibodies for electron microscopic observation Frankly, in many of these early localizations the resolution of the tissue or the specificity of the labeling left much to be desired Many of my traditional plant biochemist/physiologist colleagues said things like ‘‘I don’t believe those immunocytochemical techniques Kevin’’ One can understand this level of skepticism when organelles were not readily discernable and the label was hard to determine from background Embedding and sectioning plant tissue embedded in Lowicryl resin was very difficult and the tissue appeared extracted after prolonged embedding Only certain unique tissues such as germinating seeds were preserved sufficiently to allow for good resolution of structures However, things started to improve dramatically for plant immunocytochemistry with the introduction of the London resins These resins infiltrated plant tissues easily and could be polymerized with standard electron microscopy techniques used for epoxy-based resins The other breakthrough was the development of gold-labeled secondary antibodies Unlike ferritin, these antibodyprobes could be prepared in a variety of sizes and the preparation of the particles themselves was not difficult and they became available from numerous commercial sources as well In addition, gold probes could be used at both the light and electron microscopic levels so that a single specimen block could be used to localize at the tissue level with the light microscope and at the organelle and suborganelle level with the transmission electron microscope My goal when I entered this area was to produce micrographs that had a high level of structural preservation and a convincing immunolocalization as well When these papers started to appear in the early 1980s, I had a steady stream of visitors to the lab to learn the protocols and my laboratory phone was dubbed ‘‘the immunogold hotline’’ by my post docs in the lab! ‘‘Why don’t my localizations look like yours?’’ was the most frequent question Luckily, this is not rocket science and most of my visitors and telephone correspondents after a bit of coaching were able to localize their protein of interest A 1988 McKnight training v vi Preface class at U Georgia even resulted in a whole class full of students doing a successful electron microscopic localization even though most of the students had never performed electron microscopic studies previously Science is not done in a vacuum and certainly the development of techniques in my laboratory was heavily influenced by other plant and animal immunocytochemists Prominent among those people that were influential in these projects are Dick Trelease, J Paul Knox, John Harper, Roberto Ligrone, Andrew Staehelin, Karen Renzaglia, Tobias Baskin, and my former post-docs Andrew Bowling, John Hoffman, Timothy Sherman, Martin Vaughan, and Larry Lehnen Each of these contributed a bit of knowledge or technique that helped these experiments progress and the protocols become more refined I am also most grateful to my mentors, Martha Powell and Kenneth Stewart, in my initial training in microscopy while a graduate student at Miami University I entered graduate school planning to be a geneticist but ended up a cell biologist thanks to Martha and Ken Rex Paul, who maintained the microscopes at the Stoneville location for many years, kept the microscopes in impeccable shape and allowed a high productivity from my now retired Zeiss EM 10CR microscope with almost 38,000 micrographs produced I am also indebted to several NRI funded proposals that allowed me to hire some of the above named post docs and to develop the techniques described in this volume My retirement from the USDA in August 2010 has allowed me the time to focus more on the writing of this book, while the memories of the experiments and the many modifications we made over the years is still fresh in my mind I thank my friends Paul Knox, Andy Bowling, Dave Collings, John Harper, Roberto Ligrone, Tobias Baskin, Lacey Samuels, and Bo Kwang for supplying me with a lovely set of micrographs to help illustrate this book Immunocytochemistry, like its predecessor cytochemistry, arose out of my frustration with trying to either use a very small amount of tissue (such as that occurring in variegated chimera plants) or to determine specific reactions in a subset of that tissue using biochemical methods For example, the presence of RuBisCo in guard cell chloroplasts was the subject of much debate but immunocytochemical techniques allowed for unequivocal localizations The development of immunogold-silver and immunofluorescence on semi-thin sections for light microscopy was similarly fruitful in answering some long-standing anatomical questions Just in our lab, we have answered questions on the nature of gelatinous fibers in trees, the role of gelatinous fibers in vines, mechanisms for ballistic seed dispersal and leaf abscission As more traditional anatomists embrace these techniques, I am sure that a number of other recalcitrant questions will be answered This book is organized essentially into two sections The first chapter gives what we consider general protocols that work well on a variety of tissues and organelles, but also a number of variations that one might try in order to obtain a successful localization Most of these were developed when the more standard protocols failed The second portion of the book reviews by organelle of those techniques that may work better with that particular organelle, what unique immunocytochemical techniques can be used, and a review of some of the more Preface vii important studies on that organelle Some of the chapters also address the questions that are still outstanding and which could benefit from immunocytochemical studies My hope with the protocols outlined in this book and the description of other studies that more people will attempt these techniques and that they become more widely adopted by the plant science community Salem, Oregon Kevin Vaughn Contents Immunocytochemical Techniques Introduction Fixation Washing and Dehydration Embedding and Polymerization with the LR White LR Gold Lowicryl Resin for Embedding Samples Methacrylate Mounting, Trimming and Sectioning Collecting and Mounting Light and Transmission Electron Microscope Sections TEM Sections Light Microscopic Sections Immunocytochemical Staining Protocols Blocking Step Antibody Step Wash Steps Secondary Antibody Light Microscopy-Immunogold-Silver Fluorescence Markers Final Washes Silver Intensification Post Stains Photography and Subsequent Plate Formation TEM Photography Double Labeling Quantification and Statistics Pre-embedding Immunogold More Radical Approaches to Pre-embedding 1 10 11 12 13 16 16 17 19 19 20 23 24 24 25 25 26 27 28 29 29 30 31 35 ix 118 The Cytoskeleton Brown RC, Lemmon BE, Mullinax JB (1989) Immunofluorescent staining of microtubules in plant tissue: Improved embedding and sectioning techniques using polyethylene glycol (PEG) and Steedman’s wax Botanica Acta 102:54–61 Callarco-Gillam PD, Siebert MC, Hubble R, Mitchison T, Kirschner M (1983) Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material Cell 35:621–629 Collings DA, Wasteneys GO (2006) Actin microfilament and microtubule distribution patterns in the expanding root of Arabidopsis thaliana Can J Bot 83:579–590 Collings DA, Zsuppan G, Allen NS, Blancaflor EB (2001) Demonstration of prominent actin filaments in the root columella Planta 212:392–403 Davis FM, Tsao TY, Fowler SK, Rao PN (1983) Monoclonal antibodies to mitotic cells Proc Natl Acad Sci USA 80:2926–2930 DelVecchio AJ, Harper JDI, Vaughn KC, Baron AT, Salisbury JL, Overall RL (1997) Centrin homologues in higher plant cells are prominently associated with the developing cell plate Protoplasma 196:224–234 Doxsey SJ, Stein P, Evans L, Calarco PD, Krischer M (1994) Pericentrin, a highly conserved centrosome protein involved in microtuble organisation Cell 76:639–650 Durso NA, Cyr RJ (1994) A calcium sensitive interaction between microtubules and the higher plant homolog of elongation factor-1a Plant Cell 6:893–905 Durso NA, Leslie JD, Cyr RJ (1996) In situ immunocytochemical evidence that a homolog of protein translation elongation factor 1a is associated with microtubules in carrot cells Protoplasma 190:141–150 Dyachok J, Shao MR, Vaughn K, Bowling A, Facette M, Djakovic S, Clark L, Smith L (2008) Plasma membrane-associated SCAR complex subunits promote cortical F-actin accumulation and normal growth characteristics in Arabidopsis roots Molec Plant 1:990–1006 Faulkner CR, Blackman LM, Collings DA, Cordwell SJ, Overall RL (2009) Anti-tropomyosin antibodies co-localise with actin microfilaments and label plasmodesmata Eur J Cell Biol 88:357–369 Fisher DD, Cyr RJ (1993) Calcium levels affect the ability to immunolocalize calmodulin in cortical microtubules Plant Physiol 103:543–551 Gubler F (1989) Immunofluorescence localization of microtubules in plant root tips embedded in butyl-methyl methacrylate Cell Biol Int Rep 13:137–145 Harper JDI, Mitchison JM, Williamson RE, John PCL (1989) Does the autoimmune serum 5051 specifically recognize microtubule organizing centres in plants? Cell Biol Int Rep 13:471–483 Harper JDI, Holdaway NJ, Brecknock SL, Busby CH, Overall RL (1996) A simple and rapid technique for the immunofluorescence confocal microscopy of intact Arabidopsis root tips Cytobios 87:71–78 Hasezawa S, Nagata T (1993) Microtubule organizing centers in plant cells: Localization of a 49 kDa protein that is immunologically related to a 51 kDa protein from sea urchin centrosomes in synchronized BY-2 cells Protoplasma 176:64–74 Hepler PK (1976) The blepharoplast of Marsilea: its de novo formation and spindle production J Cell Sci 21:361–390 Hess MW, Mittermann I, Lusching C, Valenta R (1995) Immunocytochemical localization of actin and profilin in the generative cell of angiosperm pollen: TEM studies on high = pressure frozen and freeze-substituted Ledebouria socialis Roth(Hyacinthaceae) Histochem Cell Biol 104:443–451 Hoffman JC, Mullins JM (1990) A nuclear and mitotically enhanced epitope Cell Motil Cytoskel 16:68–79 Hoffman JC, Vaughn KC (1995a) Post-translational tubulin modifications in spermatogenous cells of the pteridophyte Ceratopteris richardii Protoplasma 186:169–182 Hoffman JC, Vaughn KC (1995b) Mitotic disrupter herbicides act by a single mechanism but vary in efficacy Protoplasma 186:169–182 Hoffman JC, Vaughn KC (1996) Spline and flagellar microtubules are resistant to mitotic disrupter herbicides Protoplasma 192:57–69 References 119 Hoffman JC, Vaughn KC, Joshi HC (1994) Structural and immunocytochemical characterization of microtubule organizing centers in pteridophyte spermatogenous cells Protoplasma 179:46–60 Hoffman JC, Vaughn KC, Mullins JM (1998) Fluorescence microscopy of etched methacrylate sections improves the study of mitosis in plant cells Micros Res Tech 40:369–376 Klink VP, Wolniak SM (2001) Centrin is necessary for the formation of the mitotic apparatus in spermatids of Marsilea Mol Biol Cell 12:761–776 Klink VP, Wolniak SM (2003) Changes in the abundance and distribution of conserved centrosomal, cytoskeleton and ciliary proteins during spermiogenesis in Marsilea vestita Cell Motil Cytoskel 56:57–73 Kuriyama R, Savereide P, Lefebvre P, Dasgupta S (1990) The predicted amino acid sequence of a centrosphere protein in dividing sea urchin eggs is similar to elongation factor (EF-1) J Cell Sci 95:231–236 LaClaire JW (1991) Immunolocalization of myosin in intact and wounded cells of the green alga Erodesmis vericellata (Kutzing) Borgesen Planta 184:209–217 Lambert AM (1995) Microtubule-organizing centers in higher plants: Evolving concepts Bot Acta 108:535–537 LeDizet M, Piperno G (1987) Identification of an acetylation site of Chlamydomonas alphatubulin Proc Natl Acad Sci USA 84:5720–5724 Lehnen LP, Vaughn KC (1991a) Immunofluorescence and electron microscopic investigations of the effects of dithiopyr on onion root tips Pestic Biochem Physiol 40:58–67 Lehnen LP, Vaughn KC (1991b) Immunofluorescence and electron microscopic investigations of DCPA-treated oat roots Pestic Biochem Physiol 40:47–57 Lehnen LP, Vaughn KC (1992) The herbicide sindone B affects spindle microtubule organizing centers Pestic Biochem Physiol 44:50–59 Lehnen LP, Vaughan MA, Vaughn KC (1992) Terbutol affects spindle microtubule organizing centers J Exp Bot 41:537–546 L’Hernault SW, Rosenbaum JL (1985) Chlamydomonas alpha tubulin is post-translationally modified by acetylation on the epsilon –amino of lysine Biochemistry 24:473–478 Liu B, Marc J, Joshi HC, Palevitz B (1993) A gamma tubulin related protein associated with microtubule arrays of higher plants in a cell cycle dependent manner J Cell Sci 104:1217–1228 Liu B, Joshi HC, Wilson TJ, Silflow CD, Palevitz BA, Snustad DP (1994) Gamma tubulin in Arabidopsis: Gene sequence, immunoblot and immunofluorescent studies Plant Cell 6:303–314 Marc J, Gunning BES (1986) Immunofluorescent localization of cytoskeletal tubulin and actin during spermatogenesis in Pteridium aquilimin (L.) Kuhn Protoplasma 134:163–177 Mazia D (1987) The chromosome cycle and the centrosome cycle in the mitotic cycle Int Rev Cytol 100:49–92 Miller DD, Scordilis SP, Hepler PK (1995) Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata J Cell Sci 108:2549–2563 Murata T, Sonobe S, Baskin TI, Hyodo S, Hasezawa S, Nagata T, Horio T, Hasebe M (2005) Microtubule-dependent microtubule nucleation based on recruitment of gamma tubulin in higher plants Nature Cell Biol 7:961–968 Murata T, Tanahashi T, Nishiyama T, Yamaguchi K, Hasebe M (2007) How plants organize microtubules without a centrosome J Integr Plant Biol 49:1154–1163 Myles DG, Hepler PK (1975) An ultrastuctural study of the spermatozoid of the fern, Marsilea vestita J Cell Sci 17:633–645 Oakley CE, Oakley BR (1989) Identification of gamma tubulin, a new member of the tubulin superfamily encoded by a mipA gene of Aspergillus nidulans Nature 338:662–664 Palevitz BA (1993) Morphological plasticity of the mitotic apparatus in plants and its developmental consequences Plant Cell 5:1001–1009 Radford JE, White RG (1998) Localization of a myosin-like protein to plasmodesmata Plant J 14:743–750 120 The Cytoskeleton Rajangam AS, Kumar M, Aspeborg H, Guerriero G, Arvestad L, Pansri P, Brown CJL, Hober S, Blomqvist K, Divne C, Ezcurra I, Mellerowicz E, Sundberg B, Bulone V, Teeri TT (2008) MAP20, a microtubule-associated protein in the secondary cell walls of hybrid aspen, is a target of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile Plant Physiol 148:1283–1294 Reichelt S, Knight AE, Hodge TP, Baluska F, Samaj J, Volkmann D, Kendrick-Jones J (1999) Characterization of the unconvential myosin VIII in plant cells and its localization at the postcytokinetic cell wall Plant J 19:55–567 Ryananov AG, Rudkin BB, Spirin AS (1991) Regulation of protein synthesis at the elongation stage FEBS Lett 285:170–175 Salisbury JL (1995) Centrin, centrosomes and mitotic spindle poles Curr Opin Cell Biol 130:39–45 Smeda RJ, Vaughn KC, Morrison IN (1992) A novel pattern of herbicide cross-resistance in a trifluralin-resistant biotype of green foxtail [Setaria viridis (L.) Beauv.] Pestic Biochem Physiol 42:227–241 Staiger CJ, Yuan M, Valenta R, Shaw PJ, Warn RM, Lloyd CW (1994) Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments Curr Biol 4:215–219 Staves MP, Wayne R, Leopold AC (1997) Cytochalasin D does not inhibit gravitropism in roots Am J Bot 84:1530–1535 Vandre DD, Davis FM, Rao PN, Borisy GG (1984) Phosphoproteins are components of mitotic microtubule organizing centers Proc Natl Acad Sci USA 81:4439–4443 Vandre DD, Davis FM, Rao PN, Borisy GG (1986) Distribution of cytoskeletal proteins sharing a conserved phosphorylated epitope Eur J Cell Biol 41:72–81 VanGestel K, Siegers H, von Witch M, Samaj J, Baluska F, Verbelen J (2003) Immunological evidence for the presence of homologues of the actin-related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs Protoplasma 222:45–62 Vantard M, Lambert AM, DeMey J, Picquot P, VanEldik LJ (1985) Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: Localization in the mitotic apparatus J Cell Biol 101:488–499 Vaughan MA, Vaughn KC (1987) Effects of microfilament disrupters on microfilament distribution and morphology in maize root cells Histochemistry 87:129–137 Vaughn KC (2000) Anticytoskeletal herbicides In: Nick P (ed) Plant Microtubules: Potential for Biotechnology, Springer, Berlin, pp 193–205 Vaughn KC, Harper JDI (1998) Microtubule-organizing centers and nucleating sites in land plants Int Rev Cytol 181:75–149 Vaughn KC, Bowling AJ (2008) Recovery of microtubules on the blepharoplast of Ceratopteris spermatogenous cells after oryzalin treatment Protoplasma 233:231–240 Vaughn KC, Renzaglia KS (2006) Structural and immunocytochemical characterization of the Ginkgo biloba L sperm motility apparatus Protoplasma 227:165–173 Vaughn KC, Sherman TD, Renzaglia KS (1993) A centrin homologue is a component of the multilayered structure in bryophytes and pteridophytes Protoplasma 175:58–66 Webster DR, Borisy GG (1989) Microtubules are acetylated in domains that turn over slowly J Cell Sci 92:57–65 White RG, Sack FD (1990) Actin microfilaments in presumptive statocytes of root caps and coleoptiles Am J Bot 77:17–26 White RG, Badelt K, Overall RL, Vesk M (1994) Actin associated with plasmodesmata Protoplasma 180:169–184 White RG, Barton DA (2011) The cytoskeleton in plasmodesmata: a role in intercellular transport? J Exp Bot 62:5249–5266 Wick SM (1985) The higher plant mitotic apparatus Redistribution of microtubules, calmodulin and microtubule initiating material during its establishment Cytobios 43:285–294 References 121 Wick SM, Seagull RW, Osborn M, Weber K, Gunning BES (1981) Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells J Cell Biol 89:685–690 Yokota E, McDonald AR, Liu B, Shimmen T, Palevitz BA (1995) Localization of a 170 kDa myosin heavy chain in plant cells Protoplasma 185:178–187 Yokota Y, Ueda S, Tamura K, Orii H, Uchi S, Sonobe S, Hara-Nishimura I, Shimmen T (2009) An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells J Exp Bot 60:197–212 Chapter Protein Bodies/Vacuoles and Cytoplasm Vacuoles The vacuole is a single membrane-bound organelle, which, in many cells, makes up the majority of the cell volume and has a majority of the cellular solutes The vacuole has other roles than solute storage, including compartmentalizing some of the toxic compounds (phenols and alkaloids) and also the storage of proteins in seeds Some of the early successes of the immunocytochemical localization of proteins in plants involved localization of seed storage proteins (e.g., Craig and Millerd 1981) These seed storage tissue has many advantages for this technology: it fixes very easily and is sturdy enough to embed in acrylic resins with little distortion and the proteins are present in large quantities in a single structure The large number of gold particles present over the protein bodies and the low levels of background made even the skeptical believers in the technology These storage proteins detected include cilin and legumin in peas (Craig and Millerd 1981), avenin and globulin in oats (Lending et al 1989), phaseolin and phytophemagglutin in beans (Greenwood and Chrispeels 1975), and zeins(Lending et al 1988) and calreticulin (Samaj et al 2008) in maize Because of the abundance of these proteins and the timing of their synthesis, these proteins may be tracked through the ER and Golgi vesicles before reaching the protein bodies Protein bodies in storage tissues are converted to lytic vacuoles during the germination of seeds (Bolte et al 2011) Small molecules are a prominent component of the vacuole but keeping these molecules in place after permeability barriers are broken makes their precise localization difficult Mueller and Greenwood (1978) and Vaughn and Wilson (1981) introduced caffeine (an alkaloid) to complex phenols and keep them in the vacuole during the fixation process Ferreira et al (1998) used the reverse protocol by adding tannic acid to complex the cocaine alkaloids Cocaine was found in spherical accumulations in the vacuole, made by a natural association of phenols and the alkaloid A similar sort of globule was found in the vacuoles/protein bodies in lupin seeds (Pozuelo et al 2001) K Vaughn, Immunocytochemistry of Plant Cells, DOI: 10.1007/978-94-007-6061-5_7, Ó Springer Science+Business Media Dordrecht 2013 123 124 Protein Bodies/Vacuoles and Cytoplasm Hormones Like the phenolics and alkaloids, many of the hormones are small molecules that require special fixation regimes such as carbodiimide or freeze substitution (Zavala and Brandon 1983; Dewitte and Van Ockelen 2001) Localizations of these small molecule hormones include cytokinins (Eberle et al 1987; Sossunotzov et al 1988; Ruffini-Castiglioni 1998), indole acetic acid (Ohmiya et al 1990; Ohmiya and Hayashi 1992), and abscisic acid (Sotta et al 1985; Sossountzov et al 1986, Pastor et al 1995; 1999) One of the most enigmatic photo-receptor/hormone is the protein phytochrome The localization of this compound was especially enigmatic In dark-grown seedlings, phytochrome is apparently dispersed through the cytoplasm but becomes highly sequestered after exposure to light (Pratt and Coleman 1974; Saunders et al 1983) Speculation abounded as to the organelle that was involved in this sequestration until the study of McCurdy and Pratt (1986) In that study, immunogold was used to detect phytochrome in both light and dark-grown seedlings In dark grown plants, the immunogold reaction was clearly present uniformly in the cytoplasm However, in plants exposed to light, an electron opaque deposit in the cytoplasm became the site of phytochrome localization These sites have been labeled in other systems with anti-ubiquitin antibodies, indicating these are sites are regions of ubiquination of the phytochrome before its destruction It should be noted that other workers did not find similar sites of accumulation using immunocytochemistry (Moysett et al 2001), although the tissues used were radically different Cytoplasmic Proteins Leghaemoglobin A ubiquitous component of nitrogen-fixing root nodules is the oxygen-binding protein leghaemoglobin This protein is critical in keeping oxygen concentrations low enough so that nitrogen fixation is not inhibited while at the same time allowing for normal cellular respiration (Wittenberg et al 1974) Determining the cellular localization of this protein has been attempted in a number of laboratories, and in general, the immunolocalizations have indicated a strong reaction in the cytoplasm of the infected cells but not in other cells of the nodule (Verma and Bal 1976; Robertson et al 1984; Goodchild and Miller 1997) An exception is the study of Vandenbosch and Newcomb (1988) They found about 10 % of the labeling was observed in uninfected cells However, leghaemoglobin is a relatively small molecule, and the infected and uninfected cells are connected by many plasmodesmata As the permeability barriers are broken by fixation, it is possible that some small proteins could pass from cell to cell Certainly, many cytoplasmic Cytoplasmic Proteins 125 proteins can cross the nuclear pores during fixation, and leghaemoglobin is no exception to that behavior either, with about 70–80 % of the density of the labeling found in the cytoplasm It is unlikely that leghaemoglobin has a role in the nucleus, however, but rather it is likely that this is due to artifactual movement of small molecules during the fixation and embedding process Localization of all small Mr proteins like ubiquitin are often found in the cytoplasm and the nucleus, whereas larger proteins are excluded Other Cytoplasmic Proteins Nitrate reductase has been discussed previously under chloroplast proteins as it has been falsely localized to this organelle Pre-embedding immunocytochemistry reveals a localization only in the cytoplasm and chloroplasts were unlabelled, even when the stroma was clearly exposed to the antibodies, no label was associated with this organelle (Vaughn and Campbell 1988) A very interesting localization study occurred in Triticum tauschii that had been treated with an herbicide safener (Riechers et al 2003) Traditional biochemistry had shown that the safener treatment had greatly increased the levels of a specific glutathione S-transferase and when these seedlings were examined immunocytochemically it was discovered that virtually all of the immunogold reaction was present in the epidermal cells of the coleoptiles and virtually no label in other tissues This was subsequently verified by surgically separating the leaf and coleoptiles tissues and monitoring the reactions separately; these biochemical fractionations also revealed a near exclusive localization in the coleoptiles References Bolte S, Lanquar V, Soler MN, Beebo A, Satiat-Jeunemaitre B, Bouhidel K, Thomine S (2011) Distinct lytic vacuolar compartments are embedded inside the protein storage vacuole of dry and germinating Arabidopsis thaliana seeds Plant Cell Physiol 52:1142–1152 Craig S, Millerd A (1981) Pea seed storage proteins-immunocytochemical localization with ProteinA-gold by electron microscopy Protoplasma 105:333–339 Dewitte W, Van Ockelen H (2001) Probing the distribution of plant hormones by immunocytochemistry Plant Growth Regul 33:67–74 Eberle J, Wang TL, Cook S, Wells B, Weiler EW (1987) Immunoassay and ultrastructural localization of isopentyladenine and related cytokines using monoclonal antibodies Planta 172:289–297 Ferreira JFS, Duke SO, Vaughn KC (1998) Histochemical and immunocytochemical localization of tropane alkaloids in Erythoxylum coca var coca and E novogranatense var novogranatatense Int J Plant Sci 159:492–503 Goodchild DJ, Miller C (1997) Immunogold localization of hemoglobin in Casurina root nodules Protoplasma 198:130–134 126 Protein Bodies/Vacuoles and Cytoplasm Greenwood JS, Chrispeels MJ (1975) Immunocytochemical localization of phaseolin and phytoheamagglutin in the endoplasmic reticulum and Golgi complex of developing bean cotyledons Planta 164:295–302 Lending CR, Kriz AL, Larkins BA, Bracker CE (1988) Structure of maize protein bodies and immunocytochemical localization of zeins Protoplasma 143:51–62 Lending CR, Chesnut RS, Shaw KL, Larkins BA (1989) Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L Planta 178:315–324 McCurdy DW, Pratt LH (1986) Immunogold electron microscopy of phytochrome in Avena: indentification of intracellualar sites responsible for phytochrome sequestering and enhanced pelletability J Cell Biol 103:2441–2450 Moysett L, Fernadez E, Cosrtadellas N, Simon E (2001) Intracellular localization of phytochrome in Robinia pseudoacacia pulvini Planta 213:565–574 Mueller WC, Greenwood AD (1978) The ultrastructure of phenolic storing cells fixed with caffeine J Exp Bot 29:757–764 Ohmiya A, Hayaski T, Kakiuchi T (1990) Immuno-gold localization of IAA in peach seedlings Plant Cell Physiol 31:711–715 Ohmiya A, Hayashi T (1992) Immunolocalization of IAA in leaf cells of Prunus persica at different stages of development Physiol Plant 85:439–445 Pastor A, Cortadellas N, Alegre L (1995) Immunolocalization of abscisic acid by monoclonal antibodies in Lavendula stoechas L leaves Plant Growth Regul 16:287–292 Pastor A, Lopez-Carbonell M, Alegre L (1999) Abscisic acid immunolocalization and ultrastructural changes in water-stressed lavender (Lavendula stoechas L.) plants Physiol Plant 105:272–279 Pozuelo JM, Lucas MM, de Lorenzo C, Fernandez-Pascual M, Maldonado S, de Felipe MR (2001) Immunolocalization of alkaloids and X-ray microanalysis in lupin seeds Protoplasma 218:104–111 Pratt LH, Coleman RA (1974) Phytochrome distribution in etiolated grass seedlings as assayed by an indirect antibody-labeling method Am J Bot 61:195–202 Riechers DE, Zhang Q, Xu F, Vaughn KC (2003) Tissue-specific expression and localization of the safener-induced glutathione S-transferase in Triticum tauschii Planta 217:831–840 Robertson JG, Wells B, Bisseling T, Farnden KJF, Johnson WAB (1984) Immunogold localization of leghaemoglobin in cytoplasm in nitrogen-fixing root nodules of pea Nature 311:254–256 Ruffini-Castiglioni M (1998) Immunogold 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Abscisic acid, 124 Abscission zone, 74 Acetylated tubulin, 110 AclarÒ resin, Actin, 113 Adhesive, 73 Agar, 32 AGP, 22, 67, 71 Alkaloids, 123 Anti-idiotypic antibodies, 49 Anti-tubulin, 106 Arabidopsis, 6, 7, 75, 91, 106, 115 Arabinogalactan proteins, 19, 73 Arp2/3 complex, 116 Autofluorescen, 25 Auto-immune serum, 110 Auto-immunoserum, 111 B BEEM, 8–11, 13, 14, 37, 107 Bernhard’s regressive stain, 99 Bismuth staining, 99 Blocking solution, 20 Blocking step, 19, 21 Branes, 43 BrdU, 97 BrdU; 5-bromodeoxyuridine, 97 Bundlesheath cells, 44 C Caffeine, 5, 123 Callose, 67, 69, 73, 76–79, 117 Calmodulins, 22, 36, 113 Carbodiimide, Catalase, 36, 54, 55 Cell plate, 58, 77, 78 Cell walls, 61 Cellulose, 61, 64, 76, 77, 80 Cellulose binding modules, 81 Cellulose synthase, 84, 113 Centrin, 21, 112 Chemical polymerization, Chloroplast coupling factor, 45 Chloroplast development, 47 Chloroplast lipids, 49 Chloroplast or pyrenoid , 21 Chloroplasts, 43 Chrome alum, 17 Chromocentric nucleus, 91 Confocal, 25 Cryosectioning, 32 Cryostat, 32 Cytochome b6/f complex, 46 Cytokinins, 5, 124 Cytoskeleton, 103 D DAPI, 27, 109 De-esterified homogalacturonans, 67, 70, 78 Dental wax, Detergents, 20 Diamond knife, 16, 17 Dithiothreitol (DTT), 12 DNA, 93, 97 Dodder, 71, 72 Double Labeling, 29, 30 K Vaughn, Immunocytochemistry of Plant Cells, DOI: 10.1007/978-94-007-6061-5, Ó Springer Science+Business Media Dordrecht 2013 129 130 E EGTA, Elongation factor, 113 Endochitinase, 84 Enzyme cytochemistry, EPSP synthase, 49 Ethanolic Phosphotungstic Acid, 99 Expansin, 63, 71, 74 Extensions, 72, 82 F Fern, 110 Fibers, 72, 76 Fluorescence, 24–26 Fluorescence microscopy, 27 Formaldehyde, 2, Formvar, 17 Fucosylated xyloglucan, 64 G Gamma tubulin, 111 Gelatin, 32 Gelatinous (G) fibers, 70, 72 Glutaraldehyde, 2, 3, 36 Glutathione s-transferase, 125 Glycolate oxidase, 53, 54, 56 Glyoxysome, 54 Gold grids, 16, 17 Guansonine cap, 94 Guard cells, 44 H Heat polymerization, Herbicides, 68, 77 High pressure freeze, Highly de-esterified homogalacturonans, 73, 74, 76 Highly esterified homogalacturonans, 72 Histochemistry, 1, 61 Histo-knife’, 15 Histone, 96 Homogalacturonans, 82 Hormones, 124 Hornworts, 45, 70, 110 I IAA, 2, Immunofluorescence, 1, 25 Indole acetic acid, 124 Index Intermediate filaments, 103 Isocitrate lyase, 53 L Lamin, 96 Lead acetate precipitation, 99 Lead citrate, 27 Leghaemoglobin, 124 Lignin, 63, 75 Liverworts, 79, 110 Lowicryl, 11, 12, 15, 18 LR Gold, 7, 10 LR White, 7–10, 14–16, 18, 28, 37 M Malate synthase, 53, 54 Methacrylate, 12, 18, 107, 109 Methanol, MgCl2, Microfilaments, 103 Microtome, 15 Microtubules, 103, 112 Mosses, 110 Mucilage, 73, 83 Myosins, 117 N Negatives, 29 Nickel grids, 17 Nitrate reductase, 49, 50, 125 Nogold, 24 Non-fat dried milk, 19 Nucleus, 91 O Osmium, Osmium tetroxide, P Paraformaldehyde, 105 Pectinase, 79 Pectin methylesteraseinhibitor, 84 Pectins, 75, 77 Pericentrin, 111 Periodic acid, 84 Permoun, 26 Peroxidase, 84 Peroxisomes, 53, 54, 58 Index PGA, 67 Phenols, 75, 123 Phosphotungstic acid, 85 Photo-Flo, 99 Photography, 28, 29 Phragmoplast, 112, 113 Phytochrome, 2, 124 PIPES, 3, 4, 12, 36 Plasmodesmata, 117 Pollen, 1, 5, 77 Polygalacturonic acid, 62 Poly-lysine, 18, 105 Polyphenol oxidase, 46 Pre-embedding Immunogold, 31 Profilin, 116 Protein A, 24, 25 PSII proteins, 46 Pyrenoids, 44, 46, 50 Pyroantimonate precipitation, 98 Q Quantification, 30 131 Silwet, Sodium m-periodate, 84 T Tannic acid, 5, 123 TC-2 tissue sectioner, 33 Tensin, 62 Terpenes, 75 Thylakoid, 45 Tissue sectioner, 34 Tissue-TEK Toluidine, 28 Toluidine blue, 18, 19, 24, 27, 38 Transfer cells, 68, 70 Trichomes, 75 Tropomyosin, 117 Tubulin, 106 Tween 20, U Unspecialized peroxisomes, 53 Uranyl acetate, 6, 27 Urate oxidase, 54, 55 R Rabinogalactan protein , 62 RGI, 73 Rhamnogalacturonans, 67 Rhodaminephalloidin, 113 Ribulose bisphosphate carboxylase/oxygenase (RuBisCo), 43 Ribulose bisphosphate carboxylase/oxygenase , 21 RNA, 93 Root tips cells, 75 RuBisCo activase, 45 RuBisCo, 22, 44, 45 W Wall, 21 Wash Steps, 23 Water-conducting cells, 79 Witchweed, 72 S Seed storage proteins, 1, 123 Silver, 24 Silver impregnation, 98 Silver intensification, 26 X Xylanase, 80 Xylem, 74 Xylogalacturonan, 75 Xyloglucans, 62, 67, 76, 77, 80, 82 V Vacuole, 123 Vibratome, 33, 34 Vines, 72, 73 .. .Immunocytochemistry of Plant Cells Kevin Vaughn Immunocytochemistry of Plant Cells 123 Kevin Vaughn Salem, OR USA ISBN 978-94-007-6060-8... from this list, the list of plant epitopes that have been localized is quite small Many of the protocols that are used for excellent preservation of tissues of plant cells (e.g Bozzola and Russell... herein Printed on acid-free paper Springer is part of Springer Science?Business Media (www.springer.com) Preface Immunocytochemistry of plant cells has come a long way from the first review on