Insect Histology

Dr Pedro Barbosa is a Professor Emeritus at the Department of Entomology of the University of Maryland, College Park. He has been a professor at Rutgers University, the University of Massachusetts at Amherst and at the University of Maryland. Although his research focused on the ecology of insects and insect-plant interactions, he also has an interest in insect histology. Since his retirement in 2010 he has written several books on insects. Dr Deborah Berry is Co-Director for the Histopathology and Tissue Shared Resource at the Lombardi Comprehensive Cancer Center, Georgetown University, which provides research pathology support for translational cancer research. She has over 20 years' experience in histology with a specialty in the histology of the fruit fly Drosophila melanogaster.  Dr Christina Kary is a scientific editor with the journal Genes & Development at Cold Spring Harbor Laboratory Press, New York. Before becoming an editor, she earned a PhD with Dr Eric Baehrecke at the University of Maryland and was a Jane Coffin Childs post-doctoral fellow with Dr Susan Mango at Harvard University.

Preface ix Acknowledgements xi

Introduction xiii

About the companion website xiv

1 Problems of sclerotized chitin: Softening insect cuticle 1

1.1 Introduction 1

1.2 General Methods 3

1.3 Preparations of insect eggs 14

1.4 Double Embedding Techniques 16

References 19

2 Fixation 21

2.1 Introduction 21

2.2 Aldehyde based fixatives 21

2.3 Protein denaturing 30

2.4 Picric acid based 33

2.5 Mercuric chloride based 37

2.6 SEM/TEM 40

2.7 Other 46

References 51

3 Dehydrating, clearing, and embedding 54

3.1 Dehydration 54

3.2 Clearing 60

3.3 Embedding General 65

3.4 Embedding – Ester Wax 73

3.5 Embedding – Methacrylate 74

References 77

4 Staining 79

4.1 Single-contrast staining – Carmines 81

4.2 Single contrast staining – Nuclear Stains 83

4.3 Single contrast staining – General Stains 86

4.4 Single contrast staining – Golgi 89

4.5 Single contrast staining – Eggs 89

4.6 Single contrast staining – Silver Stains 90

4.7 Polychrome staining techniques – General 92

4.8 Polychrome staining – Brain/Nerve 102

4.9 Polychrome staining – blood 103

4.10 Single contrast procedures for chitinous material 105

4.11 Polychrome staining procedures for chitinous material 106

4.12 Polychrome staining for chitinous material – KOH 110

4.13 Polychrome staining for chitinous material – Differential staining of Individual Organs 111

4.14 Staining of specific tissues 113

4.15 Two dye combinations 114

References 117

5 Immunohistochemical techniques 119

5.1 Introduction 119

5.2 General immunostaining techniques 127

5.3 Immunolabeling of samples for Transmission Electron Microscopy (TEM) 135

5.4 Proliferation assays 140

5.5 Methods to detect specific proteins 142

References 144

6 Use of genetic markers in insect histology 146

6.1 Introduction 146

6.2 Inducible genetic markers 149

6.3 Mosaic gene expression 156

6.4 Fluorescent markers for live imaging and kinetic microscopy 165

References 169

7 Fluorescence 171

7.1 Introduction 171

References 192

8 Mounting 194

8.1 Introduction 194

References 206

9 Preparation of whole mounts 208

9.1 Introduction 208

References 229

10 Preparation of whole mounts for staining 231

10.1 Introduction 231

10.2 Detection of NAPDHd 237

10.3 SEM 238

10.4 In situ hybridization 240

References 244

11 Preparation of genitalia, mouthparts and other body parts 246

References 256

12 Preparation of chromosomes 258

References 288

13 Preparation of other specific insect organs and tissues 290

13.1 Introduction 290

References 323

Appendix Dissecting fluids and saline solutions 325

Index 333