Plant Microtechnique and Microscopy

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