Plant Microtechnique and Microscopy

Paperback | January 1, 1999

bySteven E. Ruzin

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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 isdemonstrated 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 tissueprocessing, 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 deconvolutionwide-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 Microscopy also serves as an indispensable reference for all students of microscopy, histology, and histological technique. It is a valuable addition to every biological laboratory.

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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 microscop...

Steven E. Ruzin is at University of California, Berkeley.
Format:PaperbackDimensions:336 pages, 8.39 × 10.91 × 0.71 inPublished:January 1, 1999Publisher:Oxford University Press

The following ISBNs are associated with this title:

ISBN - 10:0195089561

ISBN - 13:9780195089561

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Table of Contents

INTRODUCTIONABBREVIATIONS1. QUICK STARTParaffin embedding--Histology or in situ hybridizationParaffin embedding--Microwave methodGlycol methacrylate embedding--HistologyButyl/methyl methacrylate embedding--in situ hybridization or immunolocalizationSteedman's wax embedding--ImmunolocalizationChromosome squashes--Maize meiocytesHematoxylin staining--Microwave techniqueAdjusting Kohler illumination2. MICROSCOPYMagnifiers and microscopesCompound microscopesPrinciples of specimen illuminationImparting contrast using inherent differences in index of refraction within the specimenConfocal and wide-field deconvolution microscopyObtaining microscopic measurements3. CHEMICAL FIXATIONTypes of chemical fixativesThe quality of fixationTissue storageThe mechanics of fixationCoagulating fixativesNoncoagulating fixativesRecommendations for fixing plant tissues4. TISSUE DEHYDRATIONGeneral protocolDehydration using a graded solvent series--Ethanol, acetoneRapid Dehydration5. INFILTRATING AND EMBEDDING TISSUESParaffin infiltrating and embeddingPolyethylene glycol and derivatives--Infiltration and embeddingPlastic infiltrating and embedding6. SECTIONING AND MOUNTINGGeneral stepsMounting blocks to microtome stubsFactors influencing the quality of sectioningThe microtome knifeSetting up the microtomeSectioning methodsMounting sections to glass slides7. STAININGChemical classification of dyesDye chemistryMicrotechnique stainsGeneral staining proceduresStaining equipmentSelected staining methods for plant microtechniqueStaining epoxy-embedded tissuesReactive dyesCytogenetic techniques with squashed materialCytogenetic stains for sectioned materialStaining pollen tubesMounting the coverslip8. ALTERNATE METHODS OF MICROTOMYFree-hand sectionsVibratomeFreezing microtome (cryotome, cryostat)Sliding microtome9. SPECIAL METHODSTissue clearingMacerating woody tissuesMacerating gymnosperm leaves for epidermal peelsMacerating nonwoody tissuesSectioning carbonized wood samplesMicroincinerationBleaching tissuesPreserving color in whole mounted specimensRepairing broken microscope slides10. MICROTECHNIQUE NOTES AND PROBLEM SOLVINGTissue collectionFixationFixatives for algaeTanninsHard materialsSliding microtomeClearingDehydration and infiltrationEmbeddingMicrotomyFree hand sectionsStaining11. HISTOCHEMISTRY AND CYTOCHEMISTRYThe relationship of the probe to the targetCytochemical localization of cell componentsCytochemical localization of enzymesCytochemical fluorescence microscopyFluorescent dyes for specific applicationsIntrinsic fluorescenceBackground fluorescence and photobleaching12. LOCALIZATION OF MOLECULAR TARGETS IN TISSUESImmunolocalizationExample protocols for immunolocalizationTissue printing to detect proteins and RNA in plant tissueIn situ hybridizationAutoradiography--Detecting incorporated radioactive elements in tissue sectionsWhole-mount in situ hybridizationTyramide signal amplificationTUNEL Assay for detecting DNA degradation and programmed cell deathFluorescence in situ hybridizationAPPENDICESI. ToxicsII. BuffersIII. General Information and Common CalculationsIV. The Mercury Arc Lamp in Fluorescence MicroscopyV. Manufacturers and VendorsVI. OpticsBibliographyIndex

Editorial Reviews

"Excellent resource and text for students who will be using microtechniques and microscopy. Well written and illustrated. Should be in everyone's laboratory and in the library."--David R. Dobbins, Millersville University of Pennsylvania