Plant microtechnique has generated renewed interest in recent years, due in part to the need for molecular biologists to visualize a gene or gene product in the context of the whole plant. Plant Microtechnique and Microscopy offers uniquely in-depth coverage of this reinvigorated field.
Thoroughly covering classical aspects of microscope slide preparation, it goes a step beyond all other available manuals by also documenting the theory and practice of modern applications.
The text opens with single-page "Quick Start" protocols that provide students with fundamental instructions to complete eight of the most common microtechnique protocols used today. The following sections cover the theory and practice of microtechnique. The traditional paraffin method is
demonstrated by explicit step-by-step protocols, and theoretical background is incorporated to give students the tools required to design their own experiments and to interpret existing results. In addition, modern applications such as methacrylate embedding and sectioning, microwave tissue
processing, fluorescence histochemistry, and in situ hybridization are discussed in detail. The manual also contains a definitive chapter on microscopy and describes, in both text and diagrams, the optical principles of techniques such as phase contrast and DIC as well as confocal and deconvolution
wide-field microscopy. Appendices on laboratory practice (chemical toxicities, common calculations, and buffer tables), an extensive appendix on optics and its application to the microscope, and an extensive bibliography of over 550 references are also included. Ideal for courses in plant biology,
Plant Microtechnique and Microscopyalso serves as an indispensable reference for all students of microscopy, histology, and histological technique. It is a valuable addition to every biological laboratory.
Plant microtechnique has generated renewed interest in recent years, due in part to the need for molecular biologists to visualize a gene or gene product in the context of the whole plant. Plant Microtechnique and Microscopy offers uniquely in-depth coverage of this reinvigorated field.
Thoroughly covering classical aspects of microscope slide preparation, it goes a step beyond all other available manuals by also documenting the theory and practice of modern applications.
The text opens with single-page "Quick Start" protocols that provide students with fundamental instructions to complete eight of the most common microtechnique protocols used today. The following sections cover the theory and practice of microtechnique. The traditional paraffin method is
demonstrated by explicit step-by-step protocols, and theoretical background is incorporated to give students the tools required to design their own experiments and to interpret existing results. In addition, modern applications such as methacrylate embedding and sectioning, microwave tissue
processing, fluorescence histochemistry, and in situ hybridization are discussed in detail. The manual also contains a definitive chapter on microscopy and describes, in both text and diagrams, the optical principles of techniques such as phase contrast and DIC as well as confocal and deconvolution
wide-field microscopy. Appendices on laboratory practice (chemical toxicities, common calculations, and buffer tables), an extensive appendix on optics and its application to the microscope, and an extensive bibliography of over 550 references are also included. Ideal for courses in plant biology,
Plant Microtechnique and Microscopyalso serves as an indispensable reference for all students of microscopy, histology, and histological technique. It is a valuable addition to every biological laboratory.
INTRODUCTION
ABBREVIATIONS
1. QUICK START
Paraffin embedding--Histology or in situ hybridization
Paraffin embedding--Microwave method
Glycol methacrylate embedding--Histology
Butyl/methyl methacrylate embedding--in situ hybridization or
immunolocalization
Steedman's wax embedding--Immunolocalization
Chromosome squashes--Maize meiocytes
Hematoxylin staining--Microwave technique
Adjusting Kohler illumination
2. MICROSCOPY
Magnifiers and microscopes
Compound microscopes
Principles of specimen illumination
Imparting contrast using inherent differences in index of
refraction within the specimen
Confocal and wide-field deconvolution microscopy
Obtaining microscopic measurements
3. CHEMICAL FIXATION
Types of chemical fixatives
The quality of fixation
Tissue storage
The mechanics of fixation
Coagulating fixatives
Noncoagulating fixatives
Recommendations for fixing plant tissues
4. TISSUE DEHYDRATION
General protocol
Dehydration using a graded solvent series--Ethanol, acetone
Rapid Dehydration
5. INFILTRATING AND EMBEDDING TISSUES
Paraffin infiltrating and embedding
Polyethylene glycol and derivatives--Infiltration and embedding
Plastic infiltrating and embedding
6. SECTIONING AND MOUNTING
General steps
Mounting blocks to microtome stubs
Factors influencing the quality of sectioning
The microtome knife
Setting up the microtome
Sectioning methods
Mounting sections to glass slides
7. STAINING
Chemical classification of dyes
Dye chemistry
Microtechnique stains
General staining procedures
Staining equipment
Selected staining methods for plant microtechnique
Staining epoxy-embedded tissues
Reactive dyes
Cytogenetic techniques with squashed material
Cytogenetic stains for sectioned material
Staining pollen tubes
Mounting the coverslip
8. ALTERNATE METHODS OF MICROTOMY
Free-hand sections
Vibratome
Freezing microtome (cryotome, cryostat)
Sliding microtome
9. SPECIAL METHODS
Tissue clearing
Macerating woody tissues
Macerating gymnosperm leaves for epidermal peels
Macerating nonwoody tissues
Sectioning carbonized wood samples
Microincineration
Bleaching tissues
Preserving color in whole mounted specimens
Repairing broken microscope slides
10. MICROTECHNIQUE NOTES AND PROBLEM SOLVING
Tissue collection
Fixation
Fixatives for algae
Tannins
Hard materials
Sliding microtome
Clearing
Dehydration and infiltration
Embedding
Microtomy
Free hand sections
Staining
11. HISTOCHEMISTRY AND CYTOCHEMISTRY
The relationship of the probe to the target
Cytochemical localization of cell components
Cytochemical localization of enzymes
Cytochemical fluorescence microscopy
Fluorescent dyes for specific applications
Intrinsic fluorescence
Background fluorescence and photobleaching
12. LOCALIZATION OF MOLECULAR TARGETS IN TISSUES
Immunolocalization
Example protocols for immunolocalization
Tissue printing to detect proteins and RNA in plant tissue
In situ hybridization
Autoradiography--Detecting incorporated radioactive elements in
tissue sections
Whole-mount in situ hybridization
Tyramide signal amplification
TUNEL Assay for detecting DNA degradation and programmed cell
death
Fluorescence in situ hybridization
APPENDICES
I. Toxics
II. Buffers
III. General Information and Common Calculations
IV. The Mercury Arc Lamp in Fluorescence Microscopy
V. Manufacturers and Vendors
VI. Optics
Bibliography
Index
"I am grateful to Steve Ruzin for assembling this incredibly useful collection of information in such a convenient and handy form." Plant Systematics & Evolution "an archive of expertise in the histochemistry field" Cell Biology International
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