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.

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