METHODS IN MOLECULAR BIOLOGY TM Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For other titles published in this series, go to www.springer.com/series/7651
Light Microscopy Methods and Protocols Edited by Hélio Chiarini-Garcia Laboratory of Structural Biology and Reproduction, Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil Rossana C.N. Melo Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil
Editors Hélio Chiarini-Garcia Department of Morphology Federal University of Minas Gerais Belo Horizonte, MG 31270-901, Brazil chiarini@icb.ufmg.br Rossana C.N. Melo Department of Biology Federal University of Juiz de Fora Juiz de Fora, MG 36036-900, Brazil rossana.melo@ufjf.edu.br ISSN 1064-3745 e-issn 1940-6029 ISBN 978-1-60761-949-9 e-isbn 978-1-60761-950-5 DOI 10.1007/978-1-60761-950-5 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010936902 Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)
Preface Of all scientific instruments, probably none has had more applications in the life sciences than the light microscope. Advances in microscope instrumentation, sample preparation and imaging techniques have been producing fundamental insights into the functions of cells and tissues. The protocols in Light Microscopy: Methods and Protocols cover a variety of brightfield and fluorescence microscopy-based approaches central to the study of a range of biological questions. The book provides information on how to prepare cells and tissues for microscopic investigations, including detailed staining procedures and how to analyze images and interpret results accurately. Techniques are presented in a friendly, step-by-step fashion with helpful information and useful tips. Section I covers selected applications of bright-field microscopy to the study of animal and plant biology. Section II covers the fundamental principles of fluorescence microscopy as well as its applications to multiple fields including immunology, ecology, cancer biology and cell signaling. Light Microscopy: Methods and Protocols addresses different needs of researchers, who are exploring the microscopic and intriguing world of the cell. We thank Prof. John M. Walker and the staff at Humana Press for their invitation, editorial guidance, and assistance throughout the preparation of this book for publication. We also would like to express our sincere appreciation and gratitude to the contributors for sharing their precious laboratory expertise with the microscopy research community. Hélio Chiarini-Garcia Rossana C.N. Melo v
Contents Preface... Contributors... v ix SECTION I BRIGHT-FIELD MICROSCOPY APPLICATIONS 1. Glycol Methacrylate Embedding for Improved Morphological, Morphometrical, and Immunohistochemical Investigations Under Light Microscopy: Testes as a Model... 3 Hélio Chiarini-Garcia, Gleydes Gambogi Parreira, and Fernanda R.C.L. Almeida 2. Histological Processing of Teeth and Periodontal Tissues for Light Microscopy Analysis... 19 Gerluza Aparecida Borges Silva, Adriana Moreira, and José Bento Alves 3. Large Plant Samples: How to Process for GMA Embedding?... 37 Élder Antônio Sousa Paiva, Sheila Zambello de Pinho, and Denise Maria Trombert Oliveira 4. Image Cytometry: Nuclear and Chromosomal DNA Quantification... 51 Carlos Roberto Carvalho, Wellington Ronildo Clarindo, and Isabella Santiago Abreu 5. Histological Approaches to Study Tissue Parasitism During the Experimental Trypanosoma cruzi Infection... 69 Daniela L. Fabrino, Grazielle A. Ribeiro, Lívia Teixeira, and Rossana C.N. Melo 6. Intravital Microscopy to Study Leukocyte Recruitment In Vivo... 81 Vanessa Pinho, Fernanda Matos Coelho, Gustavo Batista Menezes, and Denise Carmona Cara SECTION II FLUORESCENCE MICROSCOPY APPLICATIONS 7. Introduction to Fluorescence Microscopy... 93 Ionita C. Ghiran 8. Using the Fluorescent Styryl Dye FM1-43 to Visualize Synaptic Vesicles Exocytosis and Endocytosis in Motor Nerve Terminals... 137 Ernani Amaral, Silvia Guatimosim, and Cristina Guatimosim 9. Imaging Lipid Bodies Within Leukocytes with Different Light Microscopy Techniques... 149 Rossana C.N. Melo, Heloisa D Ávila, Patricia T. Bozza, and Peter F. Weller vii
viii Contents 10. EicosaCell An Immunofluorescent-Based Assay to Localize Newly Synthesized Eicosanoid Lipid Mediators at Intracellular Sites... 163 Christianne Bandeira-Melo, Peter F. Weller, and Patricia T. Bozza 11. Nestin-Driven Green Fluorescent Protein as an Imaging Marker for Nascent Blood Vessels in Mouse Models of Cancer... 183 Robert M. Hoffman 12. Imaging Calcium Sparks in Cardiac Myocytes... 205 Silvia Guatimosim, Cristina Guatimosim, and Long-Sheng Song 13. Light Microscopy in Aquatic Ecology: Methods for Plankton Communities Studies... 215 Maria Carolina S. Soares, Lúcia M. Lobão, Luciana O. Vidal, Natália P. Noyma, Nathan O. Barros, Simone J. Cardoso, and Fábio Roland 14. Fluorescence Immunohistochemistry in Combination with Differential Interference Contrast Microscopy for Studies of Semi-ultrathin Specimens of Epoxy Resin-Embedded Samples... 229 Shin-ichi Iwasaki and Hidekazu Aoyagi Subject Index... 241
Contributors ISABELLA SANTIAGO ABREU Laboratory of Cytogenetics and Cytometry, Department of General Biology, Federal University of Viçosa, Viçosa, MG, Brazil FERNANDA R.C.L. ALMEIDA Laboratory of Structural Biology and Reproduction, Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil JOSÉ BENTO ALVES University of Uberaba, Uberaba, MG, Brazil ERNANI AMARAL Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil HIDEKAZU AOYAGI Advanced Research Center, School of Life Dentistry at Niigata, The Nippon Dental University, Niigata, Japan CHRISTIANNE BANDEIRA-MELO Laboratory of Inflammation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil NATHAN O. BARROS Laboratory of Aquatic Ecology, Department of Biology, ICB, PATRICIA T. BOZZA Laboratory of Immunopharmacology, IOC, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil SIMONE J. CARDOSO Laboratory of Aquatic Ecology, Department of Biology, ICB, CARLOS ROBERTO CARVALHO Laboratory of Cytogenetics and Cytometry, Department of General Biology, Federal University of Viçosa, Viçosa, MG, Brazil HÉLIO CHIARINI-GARCIA Laboratory of Structural Biology and Reproduction, Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil WELLINGTON RONILDO CLARINDO Laboratory of Cytogenetics and Cytometry, Department of General Biology, Federal University of Viçosa, Viçosa, MG, Brazil FERNANDA MATOS COELHO Laboratory of Immunopharmacology, Department of Biochemistry and Immunology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil DENISE CARMONA CARA Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil HELOÍSA D ÁVILA Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil DANIELA L. FABRINO Laboratory of Cellular Biology, Department of Biology, ICB, IONITA C. GHIRAN Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA CRISTINA GUATIMOSIM Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil ix
x Contributors SILVIA GUATIMOSIM Department of Physiology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil ROBERT M. HOFFMAN AntiCancer Inc and Department of Surgery, University of California, San Diego, CA, USA SHIN-ICHI IWASAKI Advanced Research Center, School of Life Dentistry at Niigata, The Nippon Dental University, Niigata, Japan LÚCIA M. LOBÃO Laboratory of Aquatic Ecology, Department of Biology, ICB, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil ROSSANA C.N. MELO Laboratory of Cellular Biology, Department of Biology, ICB, GUSTAVO BATISTA MENEZES Laboratory of Immunopharmacology, Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil ADRIANA MOREIRA Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil NATÁLIA P. NOYMA Laboratory of Aquatic Ecology, Department of Biology, ICB, DENISE M.T. OLIVEIRA Department of Botanic, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil ÉLDER ANTÔNIO SOUSA PAIVA Department of Botanic, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil GLEYDES GAMBOGI PARREIRA Laboratory of Structural Biology and Reproduction, Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil SHEILA ZAMBELLO DEPINHO Department of Biostatistics, Institute of Biosciences, UNESP Universidade Estadual Paulista, Botucatu, SP, Brazil VANESSA PINHO Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil GRAZIELLE A. RIBEIRO Laboratory of Cellular Biology, Department of Biology, ICB, FÁBIO ROLAND Laboratory of Aquatic Ecology, Department of Biology, ICB, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil GERLUZA APARECIDA BORGES SILVA Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil MARIA CAROLINA S. SOARES Laboratory of Aquatic Ecology, Department of Biology, ICB, LONG-SHENG SONG Division of Cardiovascular Medicine, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA, USA LÍVIA TEIXEIRA Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil LUCIANA O. VIDAL Laboratory of Cellular Biology, Department of Biology, ICB, PETER F. WELLER Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA