0022-15>4/96/$3.
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The Journal of Histochemistry and Cytochemistry
Copyright 0 1996 by The Histochemical Smiety, Inc
Technical Note
I
A Differentiated Silver Intensification Procedure for the
Peroxidase-Diaminobenzidine Reaction'
BRUCE QUI"' and ANN M. GRAYBIEL
Department of Brain & Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Received for publication January 13, 1995 and in revised form July 27, 1995; accepted August 26, 1995 (5T3571).
Several silver-based procedures have been developed to in-
tensdy the peroxidase-catalyzed diaminobenzidine polymer
used as a marker in immunohistochemistry. Many of these
methods use acutely unstable reagents or reagents that are
not amenable to batch promsing. We describe a new proce-
dure with an initial treatment in dilute ammoniacal silver,
followed by a differentiation step to remove background
staining and a final toning in 0.2% gold chloride. The pro-
Introduction
Immunohistochemical analysisof neural tissue provides the potential
to reveal much of the detailed cytological structuresof neurons and
glial cells in studies both of normal neuroglial cytology and of
changes induced by aging, toxicologic insults, or degenerative dis-
ease. Such structural details are not available by methods based
on in situ hybridization studies of nucleic acid expression.
To increase the sensitivity of the immunocytochemicalprocess,
several approaches have been developed to enhance the signal
provided by the peroxidase-diaminobenzidine (DAB) reaction.
These include intensification of the DAB polymer with osmium
(I), with ferric ferrocyanide (2,3), or with heavy metals, such as nickel
or cobalt (4.5). Silver intensification procedures are more compli-
cated, but they provide very high sensitivity in enhancing the DAB
signal (6-10). We describe a novel approach to silver intensifica-
tion of the DAB signal, involving exposure to a dilute ammoniacal
silver bath, brief differentiation in dilute sodium thiosulfate, and
gold toning of the latent selective silver deposit on DAB. Prelimi-
nary results have been presented in abstract form (11).
Materials and Methods
Immunohistochemical Procedure. The silver intensification technique
presented here has been tested with similar results in immunohistochemi-
cal studies using paraformaldehyde-perfusedbrains of rat and monkey and
Supported by a fellowship from the American Parkinson Foundation
(BQ) and by the Stanley Foundation.
Correspondence to: Bruce Quinn, MD, PhD, Dept. PathologylNeu-
ropathology, NYU Medical Center, NB-4W1, 550 First Ave., New York,
NY 10016.
Vol. 44, No. 1. pp. 71-74, 1996
Printed in U S A .
cedure provides a jet-black intensification of the tan di-
aminobenzidine product, is amenable to batch processing
of free-floating sections and slide-mountede o n s , and does
not require acid-cleanedglassware. (JIiiStochemCytochem
44:71-74, 1996)
KEYWORDS:Immunoperoxidasestaining; Diaminobenzidine reac-
tion product; Immunohistochemistry; Ammoniacal silver; Rat;
Human.
autopsy tissue from human brain immersion-fixed in buffered formalin
for severalweeks. For the present study, adult rats were deeply anesthesized
and were perfused through the aorta with saline and freshly prepared 4%
paraformaldehyde in neutral 0.1 M phosphate buffer. After brief post-
fmtion, the brains were cryoprotected overnight in 20% glyceroll2% DMSO
(12) or were dehydrated through 50% and 100% ethanol (8-12 hr each)
before overnight infiltration with polyethylene glycol (13) (PEG 80%. MW
1450; PEG 20%, MW 1000) Sigma; St Louis, MO). Transverse sectionswere
cut at 20-30 pm on a sledge microtome and were stored free-floating in
PB before use. For immunostaining, sections were incubated overnight at
4°C in rabbit polyclonal anti-calretinin antiserum (Chemicon; Temecula,
CA) diluted 1:lOOO in 0.1 M PBS, 0.5% TritonX-100, and 3% normal goat
serum. Blocks from human brain were embedded in paraffin, sectioned
at 10 pm, and incubated overnight in monoclonal mouse anti-glial fibril-
lary acidic protein (GFAP) antiserum 1:SOO (Dako; Carpinteria, CA). After
washing in PBS, sectionswere incubated for 30 mincach with biotinylated
goat anti-rabbit or horse anti-mouse antibody 1:200 and the ABC Elite
streptavidin-biotin complex 1:50-1:200 (Vector Laboratories;Burlingame,
CA). The immunoperoxidasereaction was carried out with DAB 50 mgllOO
ml, H202 0.01% in 0.05 M PB for 5-10 min. Sections were photographed
in matched sets with and without silver intensification, and photography
was carried out with identical exposures and concurrent processing.
Silver Intensification Procedure
1. Floating sections or slide-mounted sections less than 10 pm thick are
thoroughly washed to remove trace salts that would precipitate the am-
moniacal silver solution. Wash three times for 10 min in high-purity
H20. Warm sections in HzO in a 56-6O'C oven.
2. Ammoniacal silver nitrate solution 0.5% is prepared according to Bar-
bosa and colleagues (14). Stock ammonium hydroxide (25-30%) is
diluted 1:l with Hz0 to facilitate titration of the dilute silver solution.
Add ammonium hydroxide drop by drop; the 0.5% silver nitrate solu-
tion will become cloudy and then clear again. Heat to 56-6O'C.
3. Incubate sections or slides for 10-12 min in ammoniacal silver in an
oven at 56-60°C.
71
72 QUI", GRAYBIEL

Figure 1. (A) Calretinin-immunoreactiveneurons in frontal cortex of rat brain, floating PEG-embedded section 30 pm thick. (E) Similar field from adjacent section
after silver intensification. Note the striking visualization of fine neuron processes. Before intensification these processes were only faintly immunostained and
were not visible at low to moderate magnifications. (C) GFAP-immunostainedsubpial astrocytes in human entorhinal cortex. The subject was a 45-year-old man.
Brain was obtained 24 hr postmortem and was immersion-fixed for several weeks before paraffin embedding and sectioning at 10 wn, (D)Adjacent section; similar
field after silver intensification, showing detailed GFAP-immunolabeled glial processes. Ears: A,B = 75 pm; C,D = 40 pm.

4. Wash for 15 sec in Hz0. 7. Tone for 2 min in 0.2% gold chloride.
5 . Wash for 15 sec in I% sodium thiosulfate (pentahydrate). 8. Wash for 15 sec in H2O.
6. Wash for 15 sec in H20. 9. Treat for 2 min in 0.5% oxalic acid.
SWER INTENSIFICATION FOR DAB
10. Wash for 15 sec in H20.
11. Treat for 5 min in 5 % sodium thiosulfate.
12. Wash thoroughly in tapwater, mount, and dehydrate and coverslip.
We found that the ammonium hydroxide must be relatively fresh (less
than 1year old). We did not find that acid-cleaned glassware was essential
for the success of this procedure, although glassware containing ammonia-
cal silver may need to be acid-cleanedafter use. The most expensive agent
is 0.2%gold chloride;floating sections and mounted sections can be toned
in small puddles of this reagent. Floating sections stiffen during the gold
toning, and folded sections should be flattened early in this step. When
used as a staining bath for slide racks or net-bottom dishes, the gold chlo-
ride could be used twice without discoloration and decomposition by in-
terposing two or three 5-7-sec water baths for each run at Step 6. (The
thiosulfate and o d i c acid solutions should not be reused.)The ammonia-
cal silver solution is stable for up to an hour at room temperature and can
be heated in aliquots for successive staining procedures. It is important
to proceed without significant pause through all steps.
Results
In unintensified sections, the cell bodies and processes of calretinin-
immunoreactiveneurons (Figure 1A) and of GFAP-immunoreactive
astrocytes (Figure IC) were immunostained with the brown DAB
reaction product. After silver intensification and gold toning, the
reaction product became black, markedly intensifying the visibil-
ity and photographic capture of fine dendrites and fine glial
processes (Figures 1B and 1D). Punctate terminal fields could be
either toned black or left tan, depending on the intensity of the
primary staining, i.e., the silver intensification appeared to pro-
duce a “step function,” so that pale nonspecific background stain-
ing or very light punctate immunostainingremained untoned. This
was a major factor contributing to the low background of the pres-
ent method. The silver intensification worked equally well on
cryoprotected frozen sections and on PEG- or paraffin-embedded
tissue. Omission of Step 5, the brief bath in 1%thiosulfate, could
result in excess background silver staining of nuclei and other struc-
tures. Delay between Steps 4 and 9 reduced or eliminated the in-
tensification, apparently because of the solubility of the initial la-
tent silver deposit and subsequent gold deposit before oxalic acid
treatment. Intensified sections have been stable for up to 5 years.
The ammoniacal silver bath with gold toning comprises a Mas-
son-Fontana stain and will blacken melanin and neuromelanin
through the argentaffin reaction.
Discussion
The method reported here, based on exposure to a very dilute am-
moniacal silver bath, represents a useful alternative to existing sil-
ver intensifications for the DAB reaction product. We originally
began experiments with this method during attempts to combine
immunostaining for tyrosine hydroxylase with Masson-Fontana
staining of occult neuromelanin in the squirrel monkey (I>),inter-
posing the present dilute ammoniacal silver bath before, after, or
between the immunostaining steps. An earlier procedure for in-
tensifying the DAB reaction product with the Bodian silver stain
for neural structures was reported by Adams in 1977 (4). Methena-
mine silver has also been used to intensify DAB (9,16).
The dilute ammoniacal silver bath of Barbosa and colleagues
73
(14) was originally chosen both for its economy and for its very low
background staining when used to stain melanin. We found that
this bath left a latent silver deposit on the DAB polymer, which
could be visualized with standard gold toning and oxalic acid steps
(17J8). Silver staining of background tissue is not suppressed but
rather is removed by differentiation in dilute thiosulfate (ref. 18,
p. 503). We found that this first thiosulfate step effectively removed
any background silver staining, including that in neuroglial nuclei
and in vascular tissue components. However, certain specific silver
deposits may remain on components stained strongly by the Mas-
son-Fontana procedure, such as silver deposits on melanin and neu-
romelanin. Sporadically, in control sections without primary anti-
body, we have noted faint blue staining of neurofibrillary tangles
and neuropil threads, but not of amyloid deposits, in paraffin sec-
tions from Alzheimer cases (data not shown). This faint staining
could be controlled in extent by prolonging the first thiosulfate
step to 20-30 sec. The Bielschowskyprocedure, which stains these
tissue components strongly, is based on a 10-20% solution of am-
moniacal silver.
The present protocol is different from the physical development
methods of Gallyas, Merchenthaler, and colleagues(for reviews see
7,19). Although highly sensitive, the Gallyas physical development
techniques, which employ a bath combining silver nitrate and for-
malin, have usually required strong acid (6,20)or metal-peroxide
(7,10,21)steps to suppress the natural argyrophilia of brain tissue.
Alternatively,high concentrationsof tungstosilicic acid have been
used to suppress background staining during physical development
(22), but this variation has been reported to compromise the sensi-
tivity of the enhancement process (8). A recent modification of
the Gallyas-Merchenthaler procedure does not have an oxidative
background suppression step but, instead, takes advantage of the
differential intensification kinetics of a nickel-DAB polymer rela-
tive to background argyrophilic staining (8). However, in each of
these methods the physical development itself, which creates the
visible silver deposit, shows a latent interval and then follows a non-
linearly accelerating rate (23), so that visual inspection of sections
is recommended for each development procedure (8). We have used
the present ammoniacal silver-gold toning protocol for several years
and have found it consistently amenable to standardized and rou-
tine batch processing of both floating and mounted sections. Kitt
et al. (24) have published a method in which immunostained sec-
tions are slide-mounted, dehydrated to xylene, rehydrated, and
stained with 2.5% silver nitrate and 2% gold chloride. The pres-
ent method uses one-fifth the silver nitrate and one-tenth the gold
chloride of the Kitt method, probably because the oxalic acid step
allows visualization of an almost occult silver-goldprecipitate. Our
use of a dilute thiosulfate differentiation step is novel among
DAB-silver intensifications and allows use of this approach in tis-
sues that otherwise show nuclear staining after silver nitrate treat-
ment, even after xylene processing (25). With the times indicated,
the method yields strong signal intensification,particularly in neu-
ronal somata, axons, and dendrites, with low background non-
specific staining.
Literature Cited
1. Johansson 0, Backman J. Enhancement of immunoperoxidase stain-
ing using osmium tetroxide. J Neurosci Methods 1983;7:185
74
2. Nemes Z.Intensification of 3,3’-diaminobenzidineprecipitation using
the ferric ferricyanide reaction, and its application to the double im-
munoperoxidase technique. Histochemistry 1987;86:415
3. Lascano EF. Berria MI. PAP labeling enhancement by osmium tetrox-
ide-potassium ferrocyanide treatment. J Histochem Cytochem 1988;
36:697
4. Adams JC. Technical considerations on the use of horseradish peroxi-
dase as a neuronal marker. Neuroscience 1977;2:141
5. Adams JC. Heavy metal intensification of DAB-based HRP reaction
product. J Histochem Cytochem 1981;29:775
6. Gallyas F, Gorcs T, Merchenthaler I. High-grade intensification of the
end-product of the diaminobenzidine reaction for peroxidase
histochemistry. J Histochem Cytochem 1982;30:183
7. Gallyas F, Merchenthaler I, Copper-H202 oxidation strikingly improves
silver intensification of the nickel-diaminobenzidine (Ni-DAB) end-
product of the peroxidase reaction. J Histochem Cytochem 1988;36:807
8. Merchenthaler I, Stankovics J, Gallyas F. A highly sensitive one-step
method for silver intensification of the nickel-diaminobenzidine end-
product of peroxidase reaction. J Histochem Cytochem 1989;37:1563
9. Rodriguez LM, Yulis R, Peruzzo B, Alvial G, Andrade R. Standardiza-
tion of various applications of methacrylate embedding and silver
methenamine for light and electron immunocytochemistry. Histo-
chemistry 1984;81:253
10. Velasco ME. The copper-peroxide-silvermethod: a highly sensitive pro-
cedure for the demonstration of Alzheimer’s disease lesions and for
signal intensificationin immunocytochemistry. Mod Path01 1992;5:455
11. Quinn B, Graybiel AM. A reliable, differentiated silver intensification
for DAB immunohistochemistry.J Histochem Cytochem 1991;39:722
12. Rosene D. Roy N, Davis B. A cryoprotection method that facilitates
cutting frozen sections of whole monkey brains for histological and
histochemical processing without freezing artifact. J Histochem
Cytochem 1986;34:1301
13. Klosen P, Maessen X, van den Bosch de Aguilar P. PEG embedding
for immunocytochemistry:application to the analysis of immunoreac-
ivity loss during histological processing. J Histochem Cytochem 1993;
41:455
QUI“, GRAYBEL.
14. Barbosa AJA, Casu0 WE Matgarida A. A simple and economicalmodifi-
cation of the Masson-Fontana method for staining melanin granules
and enterochromaffin cells. Stain Technol 1984;59:193
15. Quinn B, Graybiel AM. A novel procedure for Fontana silver impreg-
nation and immunohistochemistry on single tissue sections. Lab In-
vest 1990;62:81A
16. Scopsi L, Larsson GI. Increased sensitivity in peroxidase immunocyto-
chemistry. A comparative study of a number of peroxidase visualiza-
tion methods employing a model system. Histochemistry 1986;84:221
17. Samuel EP. Gold toning. Stain Technol 1953;28:225
18. Feigin I, Naoumenko J. Some chemical principles applicable to some
silver and gold staining methods for neuropathological studies.J Neu-
ropathol Exp Neurol 1976;35:495
19. Merchenthaler I, Gallyas F, Liposits 2. Silver-intensificationin immu-
nocytochemistry. In Bullock GR, Petrusz P, eds. Techniques in immu-
nocytochemistry. Vol 4. London: Academic Press, 1989:217
20. Merchenthaler I, Vigh S, Petrusz P,Schally AV. Immunocytochemical
localization of corticotropin releasing factor (CRF)in the rat brain. Am
J Anat 1982;165:385
21. Gallyas F, WolffJ. Metal-catalyzedoxidation renders silver intensifica-
tion selective. J Histochem Cytochem 1986;34:1667
22. Przepiorka D. Myerson D. A single-step silver enhancement method
permitting rapid diagnosis of cytomegalovirus infection in formalin-
fixed, paraffin-embedded tissue sections by in situ hybridization and
immunoperoxidase detection. J Histochem Cytochem 1986;34:1731
23. Gallyas F. Factors affecting the formation of metallic silver and the bind-
ing of silver ions by tissue components. Histochemistry 1979;64:97
24. Kitt CA, Hohmann C, CoyleJT, Price DL. Cholinergic innervation of
mouse forebrain structures. J Comp Neurol 1994;341:117
25. Lindner LE. Improvementsin the silver-stainingtechnique for nucleo-
lar organizer regions (AgNOR). J Histochem Cytochem 1993; 41:439