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Plant Microtechnique and Microscopy

OXFORD UNIVERSITY PRESS

ISBN 0-19-508956-1

BY

STEVEN E. RUZIN

BIOLOGICAL IMAGING FACILITY
COLLEGE OF NATURAL RESOURCES
UNIVERSITY OF CALIFORNIA, BERKELEY

Plant microtechnique - the preparation of microscope slides from living material - has made a resurgence due, in part, to the necessity of molecular biologists to visualize a gene or gene product in the context of the whole plant. Molecular biologists are no longer content with knowing that a gene is just being expressed; they need to know where in the organism and when during its developmental program that gene is doing what it does. This is where microtechnique comes into play. The "old fashioned" techniques of microscopy and paraffin sectioning have become indispensable. The fact that the instruction manuals are dated has resulted in this manual. This Laboratory Manual in Plant Microtechnique started from a series of handouts from my course at Berkeley in Plant Microtechnique. In the six years it took to write this text, I added background, techniques, protocols, and tables, in an effort to include most of the information a student in developmental biology requires for an anatomical and cytological investigation of plants. Finally, although the text is geared toward plant biology, it is applicable to animal biology as well.

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Contents

Introduction

Abbreviations

1 Quick Start

Paraffin embedding &endash; Histology or in situ hybridization
Paraffin embedding &endash; Microwave method
Glycol methacrylate embedding &endash; Histology
Butyl/methyl methacrylate embedding &endash; In situ hybridization or immunolocalization
Steedman's wax embedding &endash; Immunolocalization
Chromosome squashes &endash; Maize meiocytes
Hematoxylin staining &endash; Microwave technique
Adjusting Köhler illumination

 2 Microscopy

Magnifiers and microscopes
Compound microscopes
Principles of specimen illumination
Imparting contrast using inherent differences in index of refraction within the specimen
Phase Contrast Microscopy
Hoffman Modulation Contrast Microscopy
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
Fixatives for Cytology
Recommendations for fixing plant tissues

 4 Tissue dehydration

General protocol
Dehydration using a graded solvent series &endash; Ethanol, acetone
Rapid dehydration

5 Infiltrating and embedding tissues

Paraffin infiltrating and embedding
Polyethylene glycol and derivatives &endash; 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 and 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

Addendum

Steven Ruzin, Ph.D.

CNR Biological Imaging Facility
Department of Plant and Microbial Biology
University of California
Berkeley, CA 94720-3102

Wed, Aug 10, 2005