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Advances in Pathology, Microscopy & Molecular Morphology
Series Editors Jiang Gu and Gerhard W. Hacker

PUBLISHED TITLE
Gold and Silver Staining: Techniques in Molecular Morphology
Gerhard W. Hacker and Jiang Gu
Advances in Pathology, Microscopy & Molecular Morphology
Series Editors Jiang Gu and Gerhard W. Hacker

GOLD and SILVER

STAINING:
Techniques in Molecular Morphology

Edited by
GERHARD W. HACKER
Forschungsinstitut fuer Grund- und Grenzfragen
der Medizin und Biotechnologie
St. Johanns Spital, Landeskliniken Salzburg
Salzburg, Austria, EU

JIANG GU
Department of Biomedical Sciences
University of South Alabama
Mobile, Alabama

CRC PR E S S
Boca Raton London New York Washington, D.C.
 Library of Congress Cataloging-in-Publication Data

Gold and silver staining : techniques in molecular morphology / Gerhard W. Hacker,

Jiang Gu.
p. cm.
Includes bibliographical references and index.
ISBN 0-8493-1392-9 (alk. paper)
1. Immunogold labeling 2. Silver staining (Microscopy) 3. Histochemistry. I. Hacker,
Gerhard W. II. Gu, Jiang.

QR187.I482 G65 2002

611′.018—dc21 2002017541
CIP

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Printed on acid-free paper
 DEDICATION

I would like to dedicate this book to the flower of my life, my wife Ursula
Demarmels-Hacker. I thank her for her patience and encouragement during the
months of preparation of this publication, and for her unceasing interest in my
work—her smart questions are always a great source of inspiration to me.

G.W.H.

v
FOREWORD
Since time immemorial, gold and silver have fascinated humankind, whether in the
form of costly objects of art or as a means of payment. In the last century, silver has found
an application in photography and has been used in biology to visualize various tissue
phenomena in a variety of staining processes. In some of these, gold has been used to
enhance the silver reaction by heightening the contrast between the marker and the sur-
rounding tissue. Until the late 1960s, most of these silver tissue staining processes were
developed empirically. Unfortunately, the different factors in the staining processes were
not always under complete control, which led to capricious staining results. However,
advances in our knowledge of the chemical properties of silver ions have improved the
reproducibility of silver stains. Until 1983, scientists used silver ions almost exclusively in
histochemical staining, occasionally with gold chloride as an enhancing agent. Since
then, the immunogold–silver staining technique has rendered increasing sensitivity. Silver
staining techniques have also been used successfully to identify metallic elements in bio-
logical tissues.
Applications for the colloidal labels with silver intensification have extended continu-
ously to new fields and include not only cytochemistry and histochemistry but also in
situ hybridization and immunoblotting techniques. The advantages of immunogold–sil-
ver staining include its superb sensitivity and excellent contrast. However, increasing sen-
sitivity brings with it the risk of unspecific precipitations, which is why these processes
need to be carried out with great precision. Consequently, a handy presentation of the
various staining methods and up-to-date accounts of their individual advantages and
drawbacks will be a boon to researchers.
The editors of this book have made significant improvements to the immunogold–sil-
ver technique and helped popularize it for routine staining purposes. They must, there-
fore, be acknowledged for assembling in book form their own and other well-known
researchers’ expertise in the field. This will facilitate both research planning and practical
laboratory work.
Lars Grimelius, MD, Ph.D.
Departments of Genetics and Pathology
Uppsala University,
Uppsala, Sweden

vii
PREFACE
The search for highly sensitive in situ antigen and nucleotide detecting techniques
recently achieved a new milestone with the microscopic detection of single molecules.
In 1981 and 1983, two genius scientists, Gorm Danscher (Denmark) and Clive Hol-
gate (U.K.), successfully developed silver enhancement (autometallography [AMG]) and
immunogold–silver staining. The latter method uses the very small particles of colloidal
gold as the labeling marker to visualize the location of antibodies and nucleotide probes
that have been bonded to specific antigens and nucleotide sequences in tissue sections or
cytologic preparations. The initially tiny gold particles are highly uniform in size and are
enlarged further by AMG, thereby precipitating metallic silver onto the gold label surface.
Under the light microscope (LM), the enlarged particles are black and provide a highly
contrasting colored marker, standing out distinctly against the unstained background
structures. Under the electron microscope (EM), the gold or gold–silver particles are high-
ly electron-dense and provide a clear marker for target detection at the ultrastructural
level. Gold–silver staining is one of the few procedures that can demonstrate the same
target molecules at both the LM and EM levels. It is one of the most sensitive techniques
available today for low copy number antigen and nucleotide detection. This unique tech-
nique has tremendously enhanced the scientific field of molecular morphology.
The detecting sensitivity of any immunostaining or in situ hybridization technique
depends to a large degree on the signal-to-noise ratio. The larger and more visible the
labeling signals are against the same background staining, the more sensitive the tech-
nique is. The ultimate achievement of these methods is to detect a single copy of a
nucleotide sequence or a single molecule of an antigenic epitope on routinely processed
tissue sections. In combination with tyramide signal amplification and comparable
setups of label multiplication, gold–silver staining has more or less achieved this goal.
In spite of the tremendous power and potential of AMG and gold–silver staining, its
merits and applications have not been widely recognized. Different morphological labeling
methods are being used by many laboratories; however, gold–silver staining is exceptional
in many ways. The small unified sizes of the gold particle can be controlled and can facili-
tate different degrees of tissue penetration. The degree of particle enlargement by silver or
gold salt is also controllable so that to some degree the detecting sensitivity can be adjust-
ed. Gold–silver staining is easy to perform, and the reagents required are readily available.
The black color of the end product provides distinct advantages for fast screening of the
preparations; the particularity of the stain provides a very sharp and brilliant image. These
and other merits of this technique make it a favorite choice of many scientists.
The frontier of pathology is effectively moving into the age of molecular morphology.
Simple morphologic patterns, provided by routine histochemical stains alone, are no
longer adequate for depicting the subtle distinction between normal and diseased states,
that can make significant differences in prognosis and therapy. Against this background,
our goal is to provide the readers with the latest information about this extremely sensi-
tive approach to microscopically localizing minute quantities of proteins or peptides and
genetic material.
This volume gives a timely overview and detailed description of different approaches
and applications in chapters written by the leading authorities in this emerging field. The
authors include such scientists as those who made the landmark contributions to the devel-
opment of this technique. LM and EM silver staining, AMG, and heavy metal detection

ix
are described by two living legends and their collaborators, Lars Grimelius and Gorm Dan-
scher. The inventors of clustered gold covalently bound to macromolecules, Jim Hainfeld
and collaborators, describe alternative techniques of silver and gold enhancement as well as
their outstanding Nanogold® reagent and its many uses. Immunohistochemical and mol-
ecular morphological immunogold–silver staining are reviewed by Gerhard Hacker and
Raymond R. Tubbs, both of whom are long-time advocates of the technique. Its original
inventor, Osamu Fujimori, discusses his protein A–gold–silver staining method. Rick Pow-
ell presents a very promising reagent that combines a fluorescent label with gold–silver
technology. Peter Jackson, one of the original inventors of immunogold–silver staining,
introduces a new in situ hybridization method using a thermocycler designed for in situ
polymerase chain reaction (PCR). Adalberto Merighi and his outstanding group of trans-
mission electron microscopists contribute a chapter on ultrastructural applications of
immunogold–silver staining. Hajime Sawada, John M. Robinson, and coauthors describe
instructive protocols on pre- and post-embedding immuno-EM using Nanogold. Christ-
ian Schöfer and Klara Weipoltshammer, pioneers of EM tyramide signal-amplified
gold–silver staining, instruct the reader about supersensitive EM immunocytochemistry
and in situ hybridization. Finally, Bao-Le Wang and Jozef Š amaj and coworkers discuss
applications of gold–silver scanning electron microscopy in cancer research and other
fields.
The chapters in this book offer a balanced view of this emerging field. At the same time,
they enable the readers to follow step-by-step protocols to make these procedures routine
methods in their laboratories. This method allows the visualization of molecules that have
never been localized before. With it, new discoveries follow naturally.
The editors feel that far too many animals are being used for experimentation, and
very often only to fulfill legal requirements, to retest already evaluated drugs, or simply
to reach academic titles and degrees. It is the explicit wish of the editors to set an exam-
ple with this book. Responsible researchers should learn to avoid such experiments
whenever they can. Most contributors responded to our urge and used human tissue or
cell cultures in conducting experiments whenever possible. We wish to thank them for
their kindness towards our friends in the animal kingdom.
We would like to thank the many distinguished contributors to this book for sharing
their experience with the readers. Without their devotion to molecular morphology, this
title, and indeed the advancement of this exciting field, would not have been possible.
Gerhard W. Hacker
Jiang Gu

x
ABOUT THE EDITORS

Gerhard W. Hacker, Ph.D., is full professor of histochemistry, histology, and

endocrinology at Salzburg University. He received his Ph.D. in biology and biochem-
istry at the Faculty of Natural Sciences of the University of Salzburg, Austria, and stud-
ied scientific medicine at the University of London, where he received the Diploma in
Endocrinology and Pathology of the Royal Postgraduate Medical School. After further
postdoctoral training and research periods in Uppsala and Stockholm and several insti-
tutions in the U.S., he was appointed to launch a special techniques unit for diagnostics
and research at the Institute of Pathology of the Federal Hospital of Salzburg (now Lan-
deskliniken Salzburg). He now heads a research institute on basic and borderline ques-
tions of medicine and biotechnology at the St. Johanns Hospital of the Landeskliniken
Salzburg and of the University of Salzburg. His main research areas include the develop-
ment of improved laboratory technologies, such as gold-silver staining, in situ single virus
copy detection (in situ PCR), supersensitive in situ hybridization, neuropeptide research,
neuroendocrine tumors, medical ethics, and palliative care.
Jiang Gu, M.D., Ph.D., is full professor of biomedical sciences and full professor of cell
biology and neuroscience at the University of South Alabama, Mobile, AL. He obtained
his M.D. in China and was trained as a pathologist at the Beijing Medical University, Bei-
jing, China. He then obtained a Ph.D. at the Department of Pathology, Royal Postgradu-
ate Medical School, University of London. He received postdoctoral training in molecular
biology at the National Institutes of Health, Bethesda, MD. He is now co-editor-in-chief
of the journal Applied Immunohistochemistry and Molecular Morphology and has published
numerous research articles and a number of books in the field of molecular morphology.

xi
CONTRIBUTORS
Patrizia Aimar Frederic R. Furuya
Department of Veterinary Nanoprobes
Morphophysiology Yaphank, NY, USA
Neurobiology Research Group Lars Grimelius
Università degli Studi di Torino Department of Genetics and Pathology
Torino, Italy University of Uppsala
Wilhelm Barthlott Uppsala, Sweden
Botanisches Institut Thomas Grogan
University of Bonn Department of Pathology
Bonn, Germany University of Arizona School of Medicine
Elena Beltramo Tucson, AZ, USA
Department of Internal Medicine Gerhard W. Hacker
Università degli Studi di Torino Forschungsinstitut
Torino, Italy fuer Grund– und
Annie L.M. Cheung Grenzfragen der
Department of Anatomy Medizin und
University of Hong Kong Biotechnologie
Hong Kong, China St. Johanns Spital,
Gorm Danscher Landeskliniken, Salzburg
Department of Neurobiology Salzburg, Austria, EU
Institute of Anatomy James F. Hainfeld
University of Aarhus Brookhaven National Laboratory
Aarhus C, Denmark Upton, NY, USA
Hans-Jürgen Ensikat Cornelia Hauser-Kronberger
Botanisches Institut Institute of Pathology
University of Bonn Landeskliniken Salzburg
Bonn, Germany Salzburg, Austria
Michiyo Esaki Peter Jackson
Department of Anatomy Department of Histopathology and
Yokohama City University Molecular Pathology
School of Medicine The General Infirmary at Leeds
Yokohama, Japan Leeds, England, UK
Nina Flay Fraser A. Lewis
Electron Microscope Center Department of Histopathology and
Finch University of Health Sciences Molecular Pathology
The Chicago Medical School The General Infirmary at Leeds
North Chicago, IL, USA Leeds, England, UK
Osamu Fujimori Laura Lossi
Department of Anatomy Department of Veterinary
Nagoya City University Medical School Morphophysiology
Nagoya, Japan Neurobiology Research Group
Università degli Studi di Torino
Torino, Italy

xiii
Adalberto Merighi Meredin Stoltenberg
Department of Veterinary Department of Neurobiology
Morphophysiology Institute of Anatomy
Neurobiology Research Group University of Aarhus
Università degli Studi di Torino Aarhus C, Denmark
Torino, Italy Toshihiro Takizawa
James Pettay Department of Anatomy
Department of Clinical Pathology Jichi Medical School
Cleveland Clinic Foundation Tochigi, Japan
Cleveland, OH, USA Raymond R. Tubbs
Richard D. Powell Department of Clinical Pathology
Nanoprobes The Cleveland Clinic Foundation
Yaphank, NY, USA Cleveland, OH, USA
John M. Robinson Dale D. Vandré
Department of Physiology and Cell Department of Physiology and Cell
Biology Biology
Ohio State University Ohio State University
Columbus, OH, USA Columbus, OH, USA
∨
Jozef Samaj Dieter Volkmann
Institute of Plant Genetics and Botanisches Institut
Biotechnology University of Bonn
Slovak Academy of Sciences Bonn, Germany
Nitra, Slovak Republic Bao-Le Wang
Hajime Sawada Abbott Laboratories
Department of Anatomy Abbott Park, IL, USA
Yokohama City University Klara Weipoltshammer
School of Medicine Institute for Histology and Embryology
Yokohama, Japan University of Vienna
Christian Schöfer Vienna, Austria
Institute for Histology and Embryology
University of Vienna
Vienna, Austria

xiv
EDITORIAL NOTE
Inventors of Gold and Silver Staining
Gorm Danscher (Denmark) and Clive Holgate (UK) can be regarded as the two
original inventors of gold and silver staining technologies. With autometallography,
also termed silver enhancement, Danscher laid the foundation for the incredible high
sensitivity of the technique. Detection of colloidal gold (and other catalytic metals in
tissue sections) was performed first by him, whereas Holgate and colleagues had the
superb idea of combining the high amplifying potential of Danscher’s silver
precipitation reaction with immunohistochemistry. Together, they initiated a large
spectrum of the microscopic and other molecular techniques that are available today
and are based on the very same detection principle.

Prof. Gorm Danscher Prof. Clive Holgate

xv