APPLICATIONS OF IMMUNOCYTOCHEMISTRY Edited by Hesam Dehghani Applications of Immunocytochemistry Edited by Hesam Dehghani Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2012 InTech All chapters are Open Access distributed under the Creative Commons Attribution 3.0 license, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. As for readers, this license allows users to download, copy and build upon published chapters even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. Publishing Process Manager Ana Skalamera Technical Editor Teodora Smiljanic Cover Designer InTech Design Team First published February, 2012 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from orders@intechweb.org Applications of Immunocytochemistry, Edited by Hesam Dehghani p. cm. ISBN 978-953-51-0229-8 Contents Preface IX Part 1 Study of Cellular Components and Analysis of Cellular Processes by Immunocytochemistry 1 Chapter 1 Immunostaining of Voltage-Gated Ion Channels in Cell Lines and Neurons – Key Concepts and Potential Pitfalls 3 Elke Bocksteins, Andrew J. Shepherd, Durga P. Mohapatra and Dirk J. Snyders Chapter 2 Optimizing Multiple Immunostaining of Neural Tissue 17 Araceli Diez-Fraile, Nico Van Hecke, Christopher J. Guérin and Katharina D’Herde Chapter 3 Immunohistochemical Correlation of Novel Biomarkers with Neurodegeneration in Rat Models of Brain Injury 49 Shyam Gajavelli, Amade Bregy, Markus Spurlock, Daniel Diaz, Stephen Burks, Christine Bomberger, Carlos J. Bidot, Shoji Yokobori, Julio Diaz, Jose Sanchez-Chavez and Ross Bullock Chapter 4 Immunoelectron Microscopy: A Reliable Tool for the Analysis of Cellular Processes 65 Ana L. De Paul, Jorge H. Mukdsi, Juan P. Petiti, Silvina Gutiérrez, Amado A. Quintar, Cristina A. Maldonado and Alicia I. Torres Chapter 5 Immunocytochemistry of Cytoskeleton Proteins 97 Arzu Karabay, Şirin Korulu and Ayşegül Yıldız Ünal Chapter 6 Immunocytochemical Approaches to the Identification of Membrane Topology of the Na + /Cl - -Dependent Neurotransmitter Transporters 117 Chiharu Sogawa, Norio Sogawa and Shigeo Kitayama Chapter 7 Immunocytochemical Tools Reveal a New Research Field Between the Boundaries of Immunology and Reproductive Biology in Teleosts 135 Alfonsa García-Ayala and Elena Chaves-Pozo VI Contents Part 2 Specific Applications of Immunocytochemistry 159 Chapter 8 Immunocytochemistry in Early Mammalian Embryos 161 Hesam Dehghani Chapter 9 Spermiomics: A New Term Describing the Global Survey of the Overall Sperm Function by the Combined Utilization of Immunocytochemistry, Metabolomics, Proteomics and Other Classical Analytical Techniques 179 Joan E. Rodríguez-Gil Chapter 10 The Plasticity of Pancreatic Stellate Cells Could Be Involved in the Control of the Mechanisms that Govern the Neogenesis Process in the Pancreas Gland 207 Eugenia Mato, Maria Lucas, Silvia Barceló and Anna Novials Chapter 11 Application of Immunocytochemistry to Sputum Cells to Investigate Molecular Mechanisms of Airway Inflammation 231 Kittipong Maneechotesuwan and Adisak Wongkajornsilp Chapter 12 The Schwann Cell-Axon Link in Normal Condition or Neuro-Degenerative Diseases: An Immunocytochemical Approach 249 Alejandra Kun, Gonzalo Rosso, Lucía Canclini, Mariana Bresque, Carlos Romeo, Karina Cal, Aldo Calliari, Alicia Hanuz, José Roberto Sotelo-Silveira and José Roberto Sotelo Chapter 13 Immunocytochemistry of Proteases in the Study of Leishmania Physiology and Host-Parasite Interaction 267 Raquel Elisa da Silva-López Chapter 14 Immuno-Glyco-Imaging in Plant Cells: Localization of Cell Wall Carbohydrate Epitopes and Their Biosynthesizing Enzymes 297 Marie-Laure Follet-Gueye, Mollet Jean-Claude, Vicré-Gibouin Maïté, Bernard Sophie, Chevalier Laurence, Plancot Barbara, Dardelle Flavien, Ramdani Yasmina, Coimbra Silvia and Driouich Azeddine Preface Determining the expression of a given protein in specific cells, tissues, or distinct phases of cell cycle complemented by knowledge of its intracellular localization provides essential information pertinent to the biological function of the protein. Immunocytochemistry has been an invaluable tool in revealing this information. Using specific antibodies targeted to specific antigens in situ (fixed and lastingly localized in native intracellular locations), immunocytochemistry not only reveals whether a protein is expressed in a cell or a group of cells, but also discloses its intracellular localization. This technique has been employed many times in a vast array of cell biology experiments. Thus, it has experienced small modifications in different applications. This book is devoted to this technique, its application in different cellular contexts, and its adaptations. Applications of Immunocytochemistry is a collection of writings from experts in different fields of biology, organized in 14 chapters, each introducing a distinct cellular context for the application of immunocytochemistry. In addition, some of the chapters include specific technical and background information on different steps of immunocytochemistry. A table of technical contents has been included to help the reader in finding this technical information. I would like to express my sincere gratitude to the fine contribution of the authors. I also would like to thank Mrs Ana Skalamera, the Publishing Process Manager of this book, for her constant help and guidance. I hope that this book will be of great interest and use to researchers in all areas of cell biology. January 2012 Hesam Dehghani Ferdowsi University of Mashhad, Mashhad, Iran [...]... 4.2 The importance of validating your antibody One of the major difficulties of immunological detection of proteins of interest is nonspecific binding of the antibody, which causes misinterpretation of the obtained results 8 Applications of Immunocytochemistry Ensuring the lack of non-specific binding of the antibody is even more important when only a low copy number of the antigen of interest is present,... Study of Cellular Components and Analysis of Cellular Processes by Immunocytochemistry 1 Immunostaining of Voltage-Gated Ion Channels in Cell Lines and Neurons – Key Concepts and Potential Pitfalls 1Department Elke Bocksteins1,2,*, Andrew J Shepherd2,*, Durga P Mohapatra2 and Dirk J Snyders1 of Biomedical Sciences, University of Antwerp, Antwerp; of Pharmacology, University of Iowa Carver College of Medicine,... edge of the cell when the focal plane lies deeper in the cell (right image) 16 Applications of Immunocytochemistry 5.5 Bleaching and weakening of fluorescent signals A last point that has to be taken into account when using fluorescent molecules to detect proteins of interest is bleaching Fluorophores are highly light sensitive and exposure of those fluorescent molecules to light cause bleaching of. .. label and that of the protein of interest have to be cloned in frame with each other The use of genetically-encoded protein labels has a few major advantages compared to the use of immunofluorescent detection of the protein of interest Using genetically-encoded labeled proteins allows experiments to be performed in live cells, which eliminates possible artifacts that could occur as a result of cell fixation... consisting of (consecutively): the N-terminal 12 amino acid presequence of subunit IV of cytochrome c oxidase (COX4), M13 which is a 26 amino acid-long peptide derived from the calmodulin (CaM)-binding region of the skeletal muscle light-chain kinase, circularly permuted GFP (cpGFP) in which the N-and C-terminus of GFP are linked to each other, and CaM (Nagai et al., 2001) 18 Applications of Immunocytochemistry. .. emission detection channel of another fluorophore (Fig 1) This can happen in either direction, i.e Immunostaining of Voltage-Gated Ion Channels in Cell Lines and Neurons – Key Concepts and Potential Pitfalls 13 Fig 1 Bleed-through of the fluorescent signal with immunofluorescence-based detection of voltage-gated K+ (Kv) channel subunits In the upper row, the expression of Kv2.1 in a HEK293 cell has... pitfall of fluorescence-based staining is background fluorescence This could be caused by autofluorescence of the sample, non-specific binding of the antibody in the case of immunostainings and/or non-specific excitation of another fluorophore by the selected excitation wavelength when multiple fluorophores are used Such background fluorescence is a particular problem when the fluorescent signal of interest... that formaldehyde introduces does preclude the binding of antibodies to their target, some cross-links can be broken, restoring more of the native conformation to 6 Applications of Immunocytochemistry cellular structures Such processes are referred to as antigen retrieval, and are mainly enzyme or heat-mediated Incubation of specimens at temperatures of approximately 60°C in an antigen retrieval buffer... sequence therein spurred a great increase in the practicality and popularity of immunostaining; theoretically limitless quantities of * Both authors contributed equally to this work 4 Applications of Immunocytochemistry antibodies of identical specificity could be generated using cell culture This has doubtlessly accelerated the rate of discovery across all biomedical science disciplines The information... indirectly detect an antigen to which the primary antibody has bound Since the vast majority of commercial antibodies are derived from a small 10 Applications of Immunocytochemistry number of species, a wide, versatile range of secondary antibodies are available to detect any primary antibody An additional advantage of using secondary antibodies is that several secondary antibody molecules can bind to each . importance of validating your antibody One of the major difficulties of immunological detection of proteins of interest is non- specific binding of the antibody, which causes misinterpretation of the. results. Applications of Immunocytochemistry 8 Ensuring the lack of non-specific binding of the antibody is even more important when only a low copy number of the antigen of interest. APPLICATIONS OF IMMUNOCYTOCHEMISTRY Edited by Hesam Dehghani Applications of Immunocytochemistry Edited by Hesam Dehghani