Golgi-Cox Staining of Neuronal Dendrites

and Dendritic Spines With FD Rapid

GolgiStainTM Kit
Fu Du1,2
1
FD NeuroTechnologies, Inc., Columbia, Maryland
2
Corresponding author: fu.du@fdneurotech.com

The Golgi-Cox method has been one of the most effective techniques for study-
ing the morphology of neuronal dendrites and dendritic spines. However, the
reliability and time-consuming process of Golgi-Cox staining have been major
obstacles to the widespread application of this technique. To overcome these
shortcomings and to promote this invaluable technique, we developed the FD
Rapid GolgiStainTM Kit based on the principle of the methods described by
Ramón-Moliner in 1970 and Glaser and Van der Loos in 1981. The kit signifi-
cantly improves and simplifies the Golgi-Cox technique. This kit is reliable for
visualizing morphological details of neurons, allowing for analysis of various
parameters of dendritic morphology—such as dendritic length and branch-
ing pattern and dendritic spine number, shape, and size—in both animal and
postmortem human brains. A 40-min instructional video for tissue freezing,
cryosectioning, and staining is provided. 
C 2019 by John Wiley & Sons, Inc.

Keywords: dendritic morphology r dendritic spines r FD Rapid GolgiStainTM

kit r Golgi staining r Golgi-Cox

How to cite this article:

Du, F. (2019). Golgi-Cox staining of neuronal dendrites and
dendritic spines with FD Rapid GolgiStainTM Kit. Current
Protocols in Neuroscience, 88, e69. doi: 10.1002/cpns.69

For more than a century, the Golgi-Cox method has been one of the most effective BASIC
histological techniques for visualizing the morphology of neurons and their processes. PROTOCOL
The technique relies on mercury impregnation of neurons such that the fine structures
of neuronal processes, including dendrites and dendritic spines, are revealed in exquisite
detail. Labeled neurons are sparsely distributed, which allows for the complete den-
dritic arbors of individual neurons to be visualized. Although Golgi-Cox labeling is not
restricted to specific neuron types that may be identified on the basis of genetic or neu-
rochemical markers, classification of most neuron types throughout the brain is based
on original descriptions of Golgi-impregnated neurons. Labeling is sufficiently complete
that it allows analysis of various parameters of dendritic morphology—such as dendritic
length and branching pattern and dendritic spine number, shape, and size—in different
brain areas. Such analysis allows for the study of normal and abnormal morphology of
neuronal dendrites in specific neuronal subtypes in experimental animal paradigms or in
postmortem human brains.

While Golgi-Cox labeling of neurons has been useful for analysis of dendritic arborization
and dendritic spine structure, the technique has shortcomings that limit its use. The
method is complicated by the complex reagents required and the time-consuming steps,
which can lead to unpredictable reproducibility and variable labeling. To overcome

Du

Current Protocols in Neuroscience e69, Volume 88 1 of 19

Published in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpns.69

C 2019 John Wiley & Sons, Inc.
 these shortcomings, we developed the FD Rapid GolgiStainTM Kit that provides reliable
and reproducible results. This kit has been used in numerous studies (An et al., 2008;
Beggs et al., 2003; Bicanic, Hladnik, & Petanjek, 2017; Gstrein et al., 2018; Koyama,
Hattori, Nishida, Hori, & Tohyama, 2015; see Current Protocols article: Milatovic et al.,
2010; for more references, visit http://www.fdneurotech.com). The simple procedure of
this kit enables researchers with minimal histological experience to obtain consistent,
reproducible, and high-quality results. This kit can be used for processing large or small
quantities of brain samples all at once or over a period of time and works with a cryostat,
vibratome, sliding microtome, or rotary microtome.

The FD Rapid GolgiStainTM Kit was designed based on the principle of the methods
described by Ramón-Moliner (1970) and Glaser and Van der Loos (1981). This kit
is reliable for visualizing morphological details of neurons, especially dendrites and
dendritic spines, and is consistent in demonstrating their subtle changes. The kit is suitable
for the analysis of various parameters of dendritic morphology—such as dendritic length
and branching pattern and dendritic spine number, shape, and size—in different areas
of both animal and postmortem human brains. The kit can be used for processing large
or small quantities of brain samples or sections all at once or over a period of time. A
40-min instructional video for tissue freezing, cryosectioning, and staining is provided
(see Supporting Information Video 1).

The FD Rapid GolgiStainTM Kit should be used with fresh or shortly fixed brain tissue.
Using with previously formalin-fixed or fresh-frozen brains is not recommended since
satisfactory results with these types of samples are not typically obtained. Additionally,
the kit does not work on any tissue that has already been cut, either free-floating sections
or sections mounted on microscope slides.
Materials
FD Rapid GolgiStainTM Kit (FD NeuroTechnologies, cat. no. PK401) containing:
Solution A
Solution B
Solution C
Solution D
Solution E
Glass specimen retriever
Natural hair paintbrush
Dropper bottle
Plastic forceps
Animal of interest or postmortem human brain tissue
4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4; e.g., FD
NeuroTechnologies, cat. no. PF101)
Isopentane (e.g., Fisher Scientific, cat. no. O35514)
Dry ice
Cresyl violet solution (e.g., FD NeuroTechnologies, cat. no. PS102-2)
50%, 75%, 95%, and 100% ethanol
Xylene, histological grade (e.g., Pharmco)
Mounting medium (e.g., Eukitt R
Quick-Hardening Mounting medium,
Sigma-Aldrich, cat. no. 03989 or Permount R
Mounting Medium, Fisher
Scientific, cat. no. SP15)

Razor blade
Glass staining jars (e.g., DWK Life Sciences, cat. no. 900170 or 900520)

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 Plastic spoon
Cryostat (e.g., Leica Cryostat CM 1860), vibratome, sliding microtome, or rotary
microtome
Gelatin-coated microscope slides (e.g., FD NeuroTechnologies, cat. no. PO101)
Pasteur pipette
Filter paper
Glass coverslips (e.g., Thermo Fisher Scientific, cat. no. 3322)
Upright bright-field microscope with at least 10×, 40×, and 100× objective lenses

Additional reagents and equipment for rodent euthanasia (see Current Protocols
article: Donovan & Brown, 2005)
CAUTION: The kit contains reagents that are toxic and harmful when in contact with
skin or inhaled and may be fatal if ingested. Do not pipette by mouth. Avoid inhalation
and contact with skin and eyes. In case of contact, wash immediately with generous
amounts of water and seek medical attention. If swallowed, wash out mouth with water
and immediately seek emergency medical attention.

CAUTION: Perform experiment under a fume hood. Wear suitable protective clothing,
gloves, and eye/face protection while handling kit reagents. Wash hands thoroughly after
performing the experiment.

NOTE: All protocols using live animals must first be reviewed and approved by an
Institutional Animal Care and Use Committee (IACUC) or must conform to governmental
regulations regarding the care and use of laboratory animals.
Tissue preparation and sectioning
Before starting, make the following preparations: Clean and rinse all containers (plastic
preferred) with distilled water. Do not use metal implements whenever Solutions A
and B are present. Keep containers tightly closed at all times. Protect tissues treated
with Solutions A and B, including sections, from light whenever possible. Perform the
procedure at room temperature unless specifically indicated.

1. Prepare impregnation solution (Solution A/B) by mixing equal volumes of Solutions

A and B at least 24 hr prior to use and leave unstirred.
CAUTION: Solutions A and B (which contain mercuric chloride, potassium dichromate,
and potassium chromate) are toxic if they come into contact with skin and may be fatal
if swallowed. Do not breathe vapor or fumes from these solutions.
CAUTION: Do not pour the waste of Solutions A and B into the sink. Collect waste in
a separate waste container, and contact the local safety office or a licensed professional
waste disposal service to dispose of this material.
The impregnation solution should be stored at room temperature in the dark before use
and used within 4 weeks.

2. Deeply anesthetize experimental animal with IACUC-approved methods before

sacrificing.
Do not perfuse animals unless absolutely necessary. If perfusion is necessary, tissue must
NOT be postfixed (see step 3 for more details).
3. Optional: Perfuse animal with 4% paraformaldehyde in 0.1 M phosphate buffer
(pH 7.4) according to the following suggested protocol:
Perfusion with 4% paraformaldehyde prepares brains for both Golgi-Cox impregnation
and immunostaining.
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Current Protocols in Neuroscience
 a. Perfuse animal with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)
for 4 min.
b. Remove brain from skull immediately after perfusion.
c. Divide brain (e.g., mouse or rat brains) into 2 hemispheres or 5- to 10-mm thick
blocks.
d. For Golgi-Cox staining, continue to step 5. DO NOT POSTFIX TISSUE!
e. For immunostaining, continue to process tissue according to desired protocol.
4. Remove animal brain (or postmortem human brain tissue) from the skull as quickly
as possible. Be sure to handle carefully to avoid damaging or pressing the tissue.
Large brain specimens, including rat brains, should be divided with a sharp blade into
blocks of 5 to 10 mm thickness. For example, a rat brain may be divided either sagittally
into 2 hemispheres or coronally into 2 to 3 blocks depending on the regions of interest.

5. Rinse tissue quickly in double distilled or Milli-Q water to remove blood from the
surface.
6. Immerse tissue in impregnation solution (Solution A/B; from step 1), and store at
room temperature for 2 weeks in the dark. Replace impregnation solution after the
first 6 hr of immersion or on the next day. Gently swirl (do not shake!) the tissue
container side to side for a few seconds at least twice a week during the impregnation
period.
Use at least 5 ml impregnation solution for each cm3 of tissue. However, it is strongly
recommended to use 1.5 times the minimum amount of impregnation solution (e.g., for
a mouse brain, instead of using 5 ml, use 7.5 ml). Note that using a lower volume
of impregnation solution may decrease the intensity and reliability of staining. The
impregnation solution should be prepared at least 24 hr prior to use and left unstirred. It
is important to use the top part of the solution that is free of precipitate.
In most cases, 2 weeks of impregnation is satisfactory. However, variations in type and size
of tissue may require shorter or longer impregnation to obtain the best results. The optimal
time should be obtained by trial for each type of tissue, but 3 weeks should be sufficient for
most tissues. Note that prolonging the impregnation may increase background staining.

7. Transfer tissue into Solution C, and store at room temperature in the dark for at
least 72 hr (up to 1 week). Replace Solution C at least once after the first 24 hr of
immersion or on the next day.
The amount of Solution C should be at least 7 times the tissue volume. Using a lower vol-
ume of Solution C may result in higher background staining and nonspecific precipitate.

8. Freeze tissue for sectioning with a cryostat or sliding microtome. For example, to
rapidly freeze tissue:

a. Place tissue in a plastic spoon, and slowly dip into isopentane that has been
precooled with dry ice.
The temperature should be kept below −70°C, and the dipping should take about 1 min
(the slower the better).
b. After the tissue is completely immersed in isopentane, keep in the isopentane for
a few seconds, and then place on dry ice for another minute to ensure that the
tissue is well frozen.
Do not let the tissue thaw before the sections are cut. See Supporting Information Video
1 for tissue freezing.
To prevent damage to tissue caused by ice crystal formation and to preserve the best
possible cell morphology, the tissue must be properly frozen before sectioning.

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9a. Cut 100- to 200-µm sections using a cryostat (recommended) at −20°C to −23°C.
If the cryostat has only 1 temperature control, set the cryostat temperature to −23°C
at least 4 hr before cutting. If the cryostat has 2 temperature settings, set the chamber
temperature 1°C colder than that of the specimen head. In most cases, −23°C is satis-
factory; however, variations in type of cryostat and tissue may require a higher or lower
chamber temperature in order to cut sections smoothly and without shattering.

a. Mount tissue on the specimen disc with distilled water, but make sure that
the tissue does not thaw (may be done on dry ice).
Tissue may also be mounted with any type of tissue freezing medium, including op-
timal cutting temperature (OCT) compound, but avoid cutting through the freezing
medium. Do not embed the tissue in OCT. If the tissue must be embedded for cutting,
use TFMTM tissue freezing medium.
To help prevent sections from sticking to the blade, the brain surface may be coated
with a thin layer of distilled water (ice) using a paintbrush. Make sure that the brain
does not thaw at any time. The brain can be coated either before or after mounting
on the specimen disc. When coating with ice, the brain should be very cold so that
the water freezes immediately. To coat the brain before mounting on specimen disc,
first place the brain directly on dry ice. Then, dip a paintbrush in cold, deionized
water, and brush the surface of the brain to apply a thin layer of water using brush
strokes that are fast enough to avoid paintbrush hairs sticking to the brain.

b. After tissue is mounted on the specimen disc, place tissue on dry ice for 10
min so that both the tissue and specimen disc touch the dry ice.
c. Immediately install specimen disc with tissue on the cryostat, and wait 3 to
5 min before cutting 1 or 2 sections.
Do not use the anti-roll plate.

d. Set cryostat to the thickness of sections to be cut, and cut sections.

Each type of cryostat may vary, but all types should be able to cut thick sections.
If the cryostat does not have the option to set the section thickness to >60 µm,
such as for 100-µm sections (or thicker), cut sections as follows: (1) Set cryostat to
50-µm section thickness. (2) Roll cryostat counterclockwise slowly, and stop when
the tissue almost touches the blade (or knife). (3) Roll cryostat clockwise to cut
the tissue. If the tissue is too cold (e.g., sections show cracks that are parallel to
the blade), wait 1 to 2 min before the next trial. Repeat this step until high-quality
sections are achieved, and then continue cutting. (4) Repeat to cut each section.
See Supporting Information Video 1 for cryosectioning.

9b. Optional: Cut 100- to 200-µm sections with other types of microtomes, such as
vibratome, sliding microtome, or rotary microtome.
If using a vibratome, the impregnated brain should be embedded in agarose or gelatin.
However, for collecting sections, the cutting chamber must be filled with Solution C,
otherwise sections may crack upon drying.
If using a sliding microtome, both the stage and blade need to be maintained at low
temperature (<0°C).
If using a rotary microtome, tissue must be embedded in paraffin after immersion in
Solution C (step 7).

10. Mount sections on gelatin-coated microscope slides using Solution C:

a. Apply a few drops of Solution C (dropper bottle provided) onto a gelatin-coated

microscope slide.
b. Transfer sections with a glass specimen retriever (provided) onto the slide.
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Current Protocols in Neuroscience
 c. After moving each section to an ideal location with a paintbrush, remove excess
Solution C from the slide using a Pasteur pipette and a strip of filter paper if
necessary.
Any solution on the slide must be removed as much as possible; otherwise, sections may
fall off the slides during staining.
d. Allow sections to dry naturally overnight at room temperature. Do not use a fan
or hot plate.
Sections should be stained as soon as possible after drying overnight, but unstained
sections may be stored in a slide box at room temperature in the dark for up to 3 days
before staining.
To prevent sections from falling off slides, properly prepared gelatin-coated microscope
slides must be used.

Golgi staining procedure

See Supporting Information Video 1 for staining before performing the following steps.
Do not let sections dry out between any steps during the staining or while coverslipping!

11. Prepare staining solution (Solution D/E), which consists of 1 part Solution D,
1 part Solution E, and 2 parts double distilled or Milli-Q water. For example, mix
the following solutions in the order listed: 10 ml Solution D, 10 ml Solution E, and
20 ml double distilled water.
The staining solution should be prepared immediately before use and may be used for up
to 100 sections (e.g., mouse brain) per 100 ml, depending on the size of sections.
Keep the bottle and staining jar containing the staining solution covered to prevent
evaporation.

12. Rinse sections in double distilled or Milli-Q water 2 times for 4 min each rinse.
Replace distilled water after each use.
To help prevent sections from falling off slides, cold double distilled or Milli-Q water that
has been stored at 4°C may be used.

13. Place sections in the staining solution (from step 11) for 10 min.
Sections should be stained in a glass or plastic staining jar with sufficient solution. Do
not stain sections on microscope slides.
The staining solution should be stirred frequently during staining.

14. Rinse sections in double distilled or Milli-Q water 2 times for 4 min each rinse.
Replace distilled water after each use.
To help prevent sections from falling off slides, cold double distilled or Milli-Q water that
has been stored at 4°C may be used.

15. Optional: Perform cresyl violet counterstaining of Golgi impregnated sections:

Counterstaining allows not only determination of the precise localization of impregnated
neurons but also visualization of anatomic landmarks that would not be evident in a
non-counterstained section. However, some fine dendritic branches and dendritic spines
of impregnated neurons may be obscured by cresyl violet.stained cellular elements.
Do not let sections dry out between any steps described below.

a. Continue to rinse sections in double distilled or Milli-Q water 2 times for

4 min each rinse. Replace distilled water after each use.
b. Place sections in 50% ethanol for 5 min.
c. Stain sections in cresyl violet solution for 20 to 30 min.
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 Staining time may be increased or decreased depending on the desired intensity
and the concentration of cresyl violet solution used.
d. Dip sections in double distilled or Milli-Q water 3 times. Replace distilled water
after each use.
e. Dehydrate sections in sequential rinses of 50%, 75%, and 95% ethanol, 30 to 50
sec each rinse, depending on the desired intensity. Do not skip any step.
The staining intensity of cellular elements and background decreases quickly in
these solutions.
f. Continue to step 17.
16. Dehydrate sections in sequential rinses of 50%, 75%, and 95% ethanol, 4 min each
rinse. Do not skip any step.
17. Dehydrate sections in 100% ethanol 4 times for 4 min each rinse.
Incubation may be prolonged if the section thickness is >150 µm.

18. Clear sections in xylene 3 times for 4 min each rinse.

Sections may be temporarily stored in xylene for a few hours before coverslipping.

19. Coverslip sections using mounting medium.

In the author’s experience, Eukitt
R
quick-hardening mounting medium minimizes the de-
velopment of nonspecific spherical-shaped precipitates in the background after prolonged
storage and is therefore recommended for this protocol.

20. Image sections using an upright bright-field microscope. Store Golgi-stained sec-
tions at room temperature protected from light.
Golgi-stained sections should be analyzed as soon as possible, ideally within
6 months. Generally, sections can be stored at room temperature in the dark for
1 year without decrease in staining intensity.

COMMENTARY
Background Information
The Golgi method, initially called “the
black reaction” was discovered by Camillo
Golgi in 1873 (Corsi, 1987). Fifteen years
later, Cajal (1888) modified the original Golgi
method, and his improved Golgi technique was
later referred to as the “rapid Golgi method.”
It was with these techniques that, for the
first time, Golgi and Cajal revealed whole
nerve cells and their processes and have since
changed the view of the brain’s cells com-
pletely. In recognition of their contribution to
the knowledge of the nervous system, Golgi
and Cajal jointly received the 1906 Nobel
Prize in Physiology and Medicine. The break-
through they made has laid the foundation of
modern neuroscience.
Despite being developed more than
140 years ago, the Golgi techniques continue
to be used as the standard for visualizing
the morphology of neurons, especially den-
drites and dendritic spines. Although many
variations of the Golgi method have since
been introduced (for review, see Braak &
Braak, 1985; Koyama, 2013, Kang et al., 2017;
Millhouse, 1981), all Golgi methods, includ-
ing their modifications, can be classified into
two major impregnation techniques. Both of
them ultimately lead to either the precipitation
of silver chromate (the silver Golgi technique)
or to the production of metallic mercury and,
probably, complex oxides of mercury (the mer-
curial Golgi technique) in nerve tissue (Braak
& Braak, 1985).
The silver Golgi techniques
The original Golgi method is a two-step
impregnation. Thus, brain specimens are first
fixed in a solution of potassium dichromate,
and then specimens are immersed in a solu-
tion containing silver nitrate (Golgi, 1873). As
a result, a small fraction of neurons and glial
cells, including their cell bodies and numer-
ous processes, are darkly stained with silver
chromate. There are two main varieties of the
silver Golgi technique that have been utilized
extensively: the rapid Golgi method and the
Golgi-Kopsch method. Du

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Current Protocols in Neuroscience
 The classic rapid Golgi method developed
by Cajal (1888) requires primary fixation in
a solution of potassium dichromate and os-
mium tetroxide. The rapid Golgi method not
only significantly reduced the time needed for
performing the Golgi staining but has also
proved to be one of the best Golgi stain-
ing techniques for visualization of neuronal
morphology, including cell bodies, axons, den-
drites, and dendritic spines (Millhouse, 1981).
With this method, Cajal demonstrated pre-
viously unimaginable morphology in virtu-
ally every part of the nervous system (Cajal,
1909). Among the fine structures of nerve
cells that were first reported by Cajal were
the dendritic spines, which were described
as small thorns, “espinas,” that projected
from the dendrites of cerebellar Purkinje cells
(Cajal, 1888). However, major drawbacks of
the rapid Golgi method include unpredictable
reproducibility and the need for a special
and unusual fixation with expensive osmium
(Braak & Braak, 1985; Millhouse, 1981).
The Golgi-Kopsch method, also known as
the aldehyde Golgi method, was established
by Kopsch (1896). Kopsch replaced osmium
tetroxide in Cajal’s rapid Golgi method with
formaldehyde. One advantage of the Golgi-
Kopsch method is that formaldehyde-fixed
materials obtained from other experiments,
even if fixed for many years (D’Amelio, 1983;
Rosoklija et al., 2003), can still be used. This
feature is of particular importance for investi-
gations in the field of neuropathology because
most postmortem human brain samples were
fixed in formaldehyde. Also, the background
staining is much clearer in Golgi-Kopsch-
stained sections than in osmium-treated tissue.
Moreover, the Golgi-Kopsch method presum-
ably stains any region of the brain from ani-
mals or postmortem human tissue and works
equally well for very young or old brains
(Millhouse, 1981). A major disadvantage of
the Golgi-Kopsch method is that fewer cells
are impregnated compared with the other
Golgi techniques. In addition, the quality
of neuronal impregnation resulting from the
Golgi-Kopsch method is not as good as that
obtained with the rapid Golgi method.
The mercurial Golgi techniques
A significant improvement of the Golgi
technique, known as the “Golgi-Cox method,”
was made by Cox (1891). He introduced the
use of a mixture of potassium dichromate
and mercuric chloride, with potassium chro-
mate added to moderate the exceedingly acidic
Du reaction of the solution. The chemistry un-
8 of 19
derlying the Golgi-Cox staining is not yet
fully understood; the black deposit formed
under the action of alkalinization in Golgi-
Cox-impregnated nerve cells was believed to
be black mercuric sulfide and some complex
oxides of the metal (Ramón-Moliner, 1970;
Stean, 1974).
In their studies of the hippocampus, both
Cajal (1909) and Lorente de Nó (1934) ob-
tained better neuronal impregnation with the
Golgi-Cox method, compared with that ob-
tained with the original rapid Golgi technique.
The Golgi-Cox method reportedly gives reli-
able impregnation of many apparently normal
neurons with rich dendritic arborization and
darkly stains both cell bodies and dendrites
with an almost colorless background (Buell,
1982; Ramón-Moliner, 1970). Therefore, it is
generally accepted that the Golgi-Cox method
provides relatively consistent results and re-
veals a larger percentage of impregnated neu-
rons (Braak & Braak, 1985; Millhouse, 1981).
It is noteworthy that the modified Golgi-Cox
impregnation solution introduced by Glaser
and Van der Loos (1981) has significantly im-
proved the staining quality of neurons, espe-
cially dendrites and dendritic spines, and has
been widely adapted into many subsequent
studies. In addition, because of a low back-
ground, Golgi-Cox-impregnated sections can
be easily counterstained with basic dyes, such
as cresyl violet (Glaser & Van der Loos, 1981;
Ramón-Moliner, 1970). With numerous mod-
ifications and improvements, the Golgi-Cox
method has been regarded as one of the most
effective techniques for demonstrating the cell
bodies and the complete dendritic tree of cor-
tical neurons (Buell, 1982; Kang et al., 2017;
Koyama, 2013).
However, the Golgi-Cox method is consid-
ered to be more complicated than the rapid
Golgi method, and the staining is unstable and
especially capricious with staining of dendritic
spines (Koyama, 2013; Ranjan & Mallick,
2010). These drawbacks have been major ob-
stacles to the widespread application of this
technique. The FD Rapid GolgiStainTM Kit
was developed to overcome these shortcom-
ings and to promote this invaluable technique.
Critical Parameters
The following parameters are critically im-
portant in order to obtain high-quality impreg-
nation of neurons, especially dendrites and
dendritic spines.
1. Immerse tissue in the impregnation so-
lution (Solution A/B), and store at room tem-
perature for 2 weeks in the dark. Replace the
Current Protocols in Neuroscience
impregnation solution after the first 6 hr of
immersion or on the next day.
Because the impregnation solution affects
the quality of neuronal impregnation, in or-
der to obtain the best possible staining, it is
strongly recommended that the amount of im-
pregnation solution used be higher than the
minimal amount required. For example, in-
stead of using 5 ml, use 7.5 ml impregnation
solution for each cm3 of the tissue per solution
change.
2. Transfer tissue into Solution C, and store
at room temperature in the dark for at least
72 hr (up to 1 week). Replace the solution at
least once after the first 24 hr of immersion or
on the next day.
Solution C also affects the quality of neu-
ronal impregnation by removing unbound
chemicals left in the tissue to avoid nonspe-
cific precipitation and to reduce background
staining. Therefore, it is recommended that
the amount of Solution C used be higher than
the minimal amount required. For example, in-
stead of using 5 ml, use 7.5 ml of Solution C for
each cm3 of the tissue per solution change.
3. Place sections in the staining solution
(Solution D/E), which consists of 1 part Solu-
tion D, 1 part Solution E, and 2 parts double
distilled or Milli-Q water, for 10 min.
It is important that sections be stained in
a glass or plastic staining jar with enough so-
lution. Do not stain sections on microscope
slides.
4. Dehydrate sections in a series of graded
ethanol washes, and clear in xylene before cov-
erslipping sections.
Do not let sections dry out between any
steps during staining and while coverslipping!
Troubleshooting
See Table 1 for common problems en-
countered with this protocol, possible causes,
and potential solutions. See Table 2 for com-
mon questions that arise when following the
protocol.
Understanding Results
With the FD Rapid GolgiStainTM Kit, in-
vestigators should expect neuronal staining as
described below. In properly processed tissues,
such as mouse and rat brains or postmortem
human brain tissue, many well-stained neu-
rons and their dendrites are intensely stained
metallic black against an almost colorless
background (Figs. 1, 2, and 3). At high mag-
nification, dendritic spines are clearly seen on
dendrites (Fig. 4). In most cases, the initial part
Current Protocols in Neuroscience
of the axon is also stained (Figs. 1, 2, 3, and 5),
though staining is hardly followed to its site of
termination. Individual axons can be seen as
well, especially in the white matter (Fig. 1),
but no axon can be traced directly to the neu-
ron of origin or to its terminal field in 100-µm
sections, making this technique less valuable
for studying the morphological connection
between neurons. Golgi-stained neurons are
present in most areas of the brain, including the
brainstem (Fig. 6). Well-impregnated neurons
with relatively completed dendritic trees are
readily observed in the cerebral cortex (Figs.
1, 3, and 7B), the hippocampus (Fig. 2), the
striatum (Fig. 7A), and the cerebellum (Purk-
inje cells; Fig. 5). In contrast, neurons in some
deep structures of the brain, such as the tha-
lamus and the habenula, are frequently poorly
stained. This is in line with previous reports
that the thalamus and the habenula are some of
the most difficult structures to stain (Braak &
Braak, 1985; Ramón-Moliner, 1970). There-
fore, the FD Rapid GolgiStainTM Kit may be
less effective for investigating neuronal den-
drites and dendritic spines in these regions.
The phenomenon that certain types of
neurons tend to be impregnated frequently,
whereas others are rarely stained (Braak &
Braak, 1985), has not been fully understood.
At this point, it is not known whether those
frequently stained neurons are always the
same types of neurons morphologically and/or
functionally or if they are simply stained
randomly. Combining Golgi methods with
other modern techniques, such as horseradish
peroxidase (HRP) tracing (Somogyi &
Smith., 1979), immunofluorescence (Koyama
& Tohyama, 2013, Spiga et al., 2011), and
electron microscopy (Koyama, Nishida, &
Tohyama, 2013), may shed some light on this
unanswered question.
Another distinct feature of Golgi staining
techniques, including the Golgi-Cox method,
is that only a small proportion of neurons are
stained, which can be best appreciated on cre-
syl violet–counterstained sections (Fig. 7). Ac-
cording to Špaček (1989), <5% of neurons
can be impregnated in the brain. The Golgi
method’s ability to stain few neurons has made
it possible to visualize the entirety of a neuron,
especially dendrites and dendritic spines, with
unsurpassed clarity. Unfortunately, the inabil-
ity to stain more neurons with this method is a
problem for the examination of neuronal pop-
ulations and densities in any brain region. This
is the reason that the FD Rapid GolgiStainTM
Kit is not used for these types of studies.
Du
9 of 19
 Table 1 Troubleshooting Guide to Problems Commonly Encountered During Golgi Staining, Their Possible
Causes, and Potential Solutions

Problem Possible cause Solution

Frozen brain melts Cryostat temperature is Decrease chamber temperature to −23°C, and wait
inside cryostat and too warm at least 4 hr before sectioning (if cryostat has only
becomes “mushy” one temperature control)
Sections stick to blade Temperature of blade To help prevent sections from sticking to the blade,
during sectioning (or knife) is too warm coat brain surface (either beforea or after mounting
on specimen disc) with a thin layer of distilled water
(ice) using a paintbrush, but make sure that brain
does not thaw at any time. When this is done, the
brain should be very cold so that water freezes
immediately. If sections are still sticky, decrease
chamber temperature by 1°C or 2°C.
Sections crack during Temperature of tissue Increase cryostat temperature by 1°C or 2°C, and
sectioning and/or cryostat is too wait at least 1 hr before sectioning
cold
Tissue was cut too fast Cut sections slowly
Blade is not sharp Use a new blade
Impregnated tissue was Use adequate amount of Solution C, and replace
not treated thoroughly Solution C at least once
with Solution C
Sections crack upon Instead of Solution C, Do not use other types of solution to replace
drying other types of solution Solution C
were used
Sections were allowed Do not let sections dry out between staining steps
to dry out during before and during coverslipping
staining procedure or
coverslipping
Sections fall off slides Wrong type of slide Use properly prepared gelatin-coated microscope
during staining was used slides
procedure
Too much Solution C Remove Solution C left on slides as much as
was left on slides when possible when mounting sections
mounting sections
High background Amount of Use adequate amount of impregnation solution and
staining and/or impregnation solution Solution C
nonspecific precipitate and/or Solution C used
along dendritic was not adequate
branches, making an
appearance similar to
a barbed wire
Sections mounted on Do not let sections dry for
microscope slides were >3 days after mounting on microscope slides
dried for too long (e.g.,
>3 days) before
staining
Air bubbles present Sections were not Wash sections in a series of graded ethanol steps,
between coverslip and thoroughly dehydrated especially pure ethanol and xylene, for 4 min each
microscope slide in ethanol and/or change; make sure that 100% ethanol used is fresh
cleared in xylene and 200 proof
Continued

10 of 19
Current Protocols in Neuroscience
Table 1 Troubleshooting Guide to Problems Commonly Encountered During Golgi Staining, Their Possible
Causes, and Potential Solutions, continued

Problem Possible cause Solution

Amount of mounting Place adequate amount of mounting medium on
medium was not each slide (e.g., 8 to 10 drops per slide, depending
adequate on number of sections)
Coverslipping was not Apply coverslip properly and slowly (may require
done properly practice in order to coverslip sections well)
Staining fades within Improper type of Use previously tested mounting medium, such as
24 hr after mounting medium used Eukitt
R
quick-hardening mounting medium or
coverslipping PermountR
mounting medium
Too many neurons Sections too thick (e.g., Cut thinner sections (e.g.,
stained in >100 µm) 100 µm, but not <80 µm)
hippocampus, making
reconstructing
neurons difficult
Many stained neurons Sections too thin (e.g., Increase section thickness if <100 µm
have short dendrites <100 µm)
Sections cut at an Make sure to cut sections at correct angle. For
incorrect angle example, when cutting coronal sections, the plane of
the sections should be perpendicular to the long axis
of the brain.
a An example protocol of how to coat the brain before mounting on the specimen disc: (1) place brain directly on dry ice; (2) dip a paintbrush

in cold deionized water; and (3) brush surface of the brain to apply a thin layer of water with brush strokes that are fast enough to avoid
paintbrush hairs from sticking to the brain.

Table 2 Frequently Asked Questions About the FD Rapid GolgiStainTM Kit or Protocol and Their
Answers

Question Answer
Can the kit be used after the Generally, the kit can still be used within 3 months after the
expiration date? expiration date.
How many mouse brains can A single kit may be used for processing up to 50 mouse
be stained with one kit? brains based on a minimum amount of each solution used.
However, it is recommended to use 1.5 times the minimum
amount of each solution used (e.g., for a mouse brain,
instead of using 5 ml, use 7.5 ml).
Can the kit be used for cell The kit has been used for cultured cells (see Koyama &
culture work? Tohyama, 2013).
Can the kit be used on The kit does not work on previously cut sections.
100-µm archival precut
human cryosections?
Is the kit suitable for staining The kit can be used for staining neurons in embryonic
embryonic mouse brains mouse brains as early as E18. To date, there is no
(e.g., E17)? information regarding whether it is suitable for staining E17
mouse brains.
Is there any benefit to No. Perfusion does not aid neuronal impregnation. Unless
perfusing animals (i.e., to tissue will also be used for other purposes, do not perfuse
reduced background)? animal with any solution.
Continued

11 of 19
Current Protocols in Neuroscience
 Table 2 Frequently Asked Questions About the FD Rapid GolgiStainTM Kit or Protocol and Their
Answers, Continued
Question
If I need to perfuse the
animal, could I perfuse with
saline prior to perfusion with
4% paraformaldehyde, or
must paraformaldehyde be
used right away?
For Rhesus monkey brains,
what is the most suitable
perfusion time for fixing the
brains with 4%
paraformaldehyde?
Can metal instruments be
used to remove the brain?
Is there any additional
information or suggestions
for staining the inner parts of
the brain, such as the
striatum?
Does a mouse brain need to
be divided before
impregnation?
Is it required that whole rat
brains be blocked before
impregnation?
Will thinner specimens
provide better result?
How much impregnation
solution is recommended for
impregnation of a whole
mouse brain? A whole rat
brain?
Is a 2-week impregnation
adequate for a whole mouse
brain, or is 3 weeks
recommended?
What is the volume of
Solution C needed for a
mouse brain?
12 of 19
Answer
Do not perfuse with saline. Perfusion with 4%
paraformaldehyde should not last for more than 4 min. Do
not postfix the brain.
Although the kit has never been tested with monkey brain, it
is believed that perfusion with 4% paraformaldehyde for
more than 4 min would likely affect the quality of
Golgi-Cox staining. Moreover, removing a monkey brain
from the skull will take much longer, so the brain may be
fixed much longer. This would probably cause more
problems even if the perfusion time is <5 min. Therefore,
perfusion of a monkey with paraformaldehyde for
Golgi-Cox staining is not recommended with this kit.
Yes. Metal tools can be used to remove the brain from the
skull as long as the tool does not touch the impregnation
solution.
For deep structures, including the striatum, the brain should
be divided into 5-mm-thick blocks. For example, a mouse
brain may be divided either coronally into 2 to 3 blocks or
sagittally into 2 hemispheres before impregnation. Use an
adequate amount of impregnation solution and Solution C
for each solution change.
This depends on the brain regions of interest. For deep
structures, such as the striatum and thalamus, the brain
should be divided either sagittally into 2 hemispheres or
coronally into 5 mm thick blocks.
Because of the size, a rat brain must be blocked either
coronally or sagittally before impregnation. Otherwise,
many structures, including the hippocampus, may not be
stained well.
Not always. Generally, 5- to 10-mm-thick specimens are
optimal, depending on the brain area of interest.
For each mouse brain, use 7.5 ml impregnation solution for
each solution change, and make sure to replace the solution
after 6 hr or on the next day. For each rat brain, use 12 to
15 ml, depending on the size of the brain. Change the
solution after 6 hr or on the next day, and make sure that the
brain is divided into 3 portions because of its size (e.g., a
coronal cut just before the hippocampus and between the
cerebrum and the brainstem).
Generally, 2 weeks are adequate.
The volume of Solution C should be at least 7 times the
volume of the tissue. Using 7.5 ml per mouse brain for each
change is highly recommended.
Current Protocols in Neuroscience
Table 2 Frequently Asked Questions About the FD Rapid GolgiStainTM Kit or Protocol and Their
Answers, Continued
Question
Can brain samples be stored
in Solution C for 2 months in
the dark at room temperature?
Does it harm the staining if
the tissue is incubated in
Solution C beyond 7 days?
If brains cannot be
immediately sectioned after
48 hr in Solution C, what is
the best way to store the
brains before sectioning?
Is tissue freezing medium
(TFM) necessary, or can
optimal cutting temperature
(OCT) medium be used
instead?
Can brains frozen in TFM be
stored at −80°C or −20°C
for a few days?
Can brains be stored at
−80°C before sectioning?
Can liquid nitrogen be used
instead of isopentane for
freezing brains before
cyrosectioning?
Which method is best for
brain sectioning?
Can Solution C be skipped to
go straight into staining?
Can a steel knife be used for
sectioning?
Can the brain be mounted on
the stage without embedding?
Will Solution C damage a
vibratome?
Is freezing the brains
necessary if cutting with a
vibratome?
Can slow freezing at −80°C
be used?
If cutting with a vibratome,
can sections be stored in
Solution C or PBS before
processing?
Current Protocols in Neuroscience
Answer
Storing impregnated brains in Solution C for 2 months is too
long, as prolonged storage may decrease staining intensity.
Brains should not be stored in Solution C for more than
10 days to avoid any potential decrease in staining intensity.
Impregnated brains can be kept in Solution C for 10 days.
However, for longer storage, freeze the brains, and store at
−80°C for up to 1 year.
Use of TFM is not necessary. If OCT is used to embed
brains or mount brains on the specimen disc, trim OCT off
as much as possible before cutting through tissue, otherwise
OCT may damage the tissue sections.
Frozen brains can be stored at −80°C for up to 1 year but
cannot be stored at −20°C or the tissue will be destroyed.
Impregnated brains can be stored at −80°C only after
immersing tissue in Solution C for at least 72 hr.
Tissue can be frozen in liquid nitrogen as long as the tissue
does not crack. If the brain is dropped in liquid nitrogen, it
will most likely crack.
Cutting impregnated brains with a cryostat is recommended.
Impregnated tissue must be treated with Solution C. Do not
skip or replace Solution C with any other solution.
Yes. A steel knife or blade can be used to cut the
impregnated brains as long as the brains have been treated
with Solution C.
Yes. It is better not to embed the brain in medium, but make
sure to mount the brain well so that it does not fall off when
cutting.
Solution C does not damage the vibratome, but be sure to
wash the chamber thoroughly with water after use, as
Solution C may have been contaminated with reagents left
in the tissue.
Freezing the brain is not necessary if cutting with a
vibratome.
The brain should NOT be slowly frozen at −80°C. This can
permanently damage the brain due to ice crystal formation.
Free-floating impregnated sections can be stored in Solution
C at −20°C for 2 weeks, but they should not be stored in
PBS. Sections should always be stained as soon as possible.
13 of 19
 Table 2 Frequently Asked Questions About the FD Rapid GolgiStainTM Kit or Protocol and Their
Answers, Continued
Question
Is it necessary to cut sections
at 80- to 240-µm thickness?
Can positively charged slides
be used instead of gelatin-
coated microscope slides?
Can gelatin-coated slides be
substituted with aminosilane-
coated slides?
How thick should mouse
brain sections be cut?
Can mounted sections be
stored at 4°C or below for a
long time? How can sections
be stored for longer periods
of time?
Is it possible to stain fewer
neurons or glia?
When should sections be
stained after cutting?
Does processing,
dehydration, and clearing
need to be performed in a
dark room?
Can any of the solutions in
the kit be reused?
After staining, how soon can
stained sections be analyzed?
How long will staining be
preserved?
Can DPX mounting medium
be used for coverslipping?
Which kind of microscope
should be used to observe
dendrites and dendritic
spines?
Is it dangerous to heat or to
microwave the mixed
Solution A and B?
14 of 19
Answer
No. Sections can be cut thicker than 240 µm, but it is
recommend to not cut sections <80 µm.
No. Properly prepared gelatin-coated slides must be used to
prevent sections from falling off during staining.
Aminosilane-coated slides have not been tested. They may
be used as long as sections do not fall off slides during
staining.
Sections of 100-µm thickness are generally adequate, but it
also depends on the purpose of the study.
No. Mounted sections should be stored at room temperature
in the dark, not in a refrigerator, before staining. Sections
should be stained within 3 days after cutting. Storage of
unstained, mounted slides for >3 days may cause
nonspecific precipitate along the dendrites.
To date, there is no effective method to limit the number of
stained neurons and glia. However, thinner sections (e.g.,
100 µm) show fewer stained neurons compared with thicker
sections (e.g., 200 µm).
Sections should be stained as soon as possible after drying
overnight or within 3 days after cutting.
No, but sections should be protected from light whenever
possible.
Generally, no solution included in the kit should be reused.
However, Solution C can be reused only for collecting
sections (filling the chamber) if using a vibratome to cut
brains. Make sure to rinse sections in fresh Solution C
before mounting on slides.
Golgi-stained sections should be analyzed as soon as
possible but ideally within 6 months. Generally, sections
can be stored at room temperature in the dark for up to
1 year without a decrease in staining intensity.
DPX mounting medium may cause fading of Golgi staining.
To observe dendrites and dendritic spines, an upright
bright-field microscope with high-powered objective lenses
(e.g., 100× oil-immersion lens) is recommended.
Solutions A and B are toxic to the skin and may be fatal if
swallowed. Heating or microwaving these solutions
produces toxic vapor or fumes that are harmful to breathe.
Therefore, Solutions A and B should not be heated or
microwaved under any circumstances.
Current Protocols in Neuroscience
Figure 1 (A) Coronal section (100 µm) obtained using a cryostat through the mouse cerebral
cortex. Note intensely stained neuronal perikarya and their processes. Arrows indicate the axons
in the white matter. (B) Low-power micrograph of an area in the mouse cerebral cortex showing
well-impregnated neurons in layers II and III of the cerebral cortex. Arrows indicate the axons.

Figure 2 Coronal section (100 µm) obtained using a cryostat through the (A) mouse hippocam-
pus and (B) rat hippocampus. Note well-impregnated pyramidal neurons in CA1 and granule cells
in the dentate gyrus. Arrows indicate the axons of granule cells.

Time Considerations solution must be prepared at least 24 hr before

Time estimates described are based on per-
forming Golgi-Cox staining with the FD Rapid
GolgiStainTM Kit by an experienced histology
technician.
Preparing materials required but not in-
cluded in the kit takes 1 hr. Tissue preparation
requires 3.25 hr per brain (or 25.3 hr for
10 brains). Mixing Solution A and Solution
B (impregnation solution) takes 10 min. It
is important to note that the impregnation
use. Sacrificing the animal (e.g., mouse),
including removing the brain from the skull
and immersing the brain in impregnation so-
lution, requires 20 min per brain (or 3.3 hr for
10 brains). Replacing impregnation solution
and Solution C, including gently swirling the
container containing the brain, requires 15
min per brain (or 30 min for 10 brains).
Freezing brains for cryosectioning, includ-
ing preparing cold isopentane, requires 30 min Du

15 of 19
Current Protocols in Neuroscience
 Figure 3 Coronal section (180 µm) obtained using a cryostat through the cerebral cortex of a
postmortem human brain. Note well-impregnated neurons with their numerous dendrites. Arrow
indicates the axon.

Figure 4 High-power micrograph of Golgi-impregnated dendrites in the mouse cerebral cortex.

Note many dendritic spines of various shapes and sizes on the dendrite (A) and on all branches
of a dendrite (B).

per brain (or 1.3 hr for 10 brains). Note that
frozen brains may be stored at −80°C for up
to 1 year before sectioning. Cryosectioning,
including mounting up to 30 sections on 10
microscope slides takes 2 hr per mouse brain
(or 20 hr for 10 brains). Note that sections
mounted on microscope slides must be stained
within 3 days. However, free-floating sections
may be stored in Solution C at −20°C for
up to 2 weeks before mounting on slides. For
safety, a researcher who has no cryosectioning
experience must be trained by a staff member
Du who has significant experience in cryosection-
ing before performing this job and may need
some practice in order to obtain high-quality
sections.
Staining takes 2.25 hr per mouse brain (or
5.6 hr for 10 mouse brains). This includes
20 min for preparing staining jars and reagents,
10 min for rinsing in water, 20 min for stain-
ing in Solution D + E (including mixing
solution and distilled water), 10 min for rins-
ing in water, 35 min for dehydrating in a se-
ries of graded ethanol rinses, and 30 min for
clearing in xylene and coverslipping (for up
to 10 slides or 40 sections). Sections may be

16 of 19
Current Protocols in Neuroscience
Figure 5 (A) Sagittal section (100 µm) obtained using a cryostat through the mouse cerebellum. Note well-
impregnated Purkinje cells. Arrows indicate the axons of Purkinje cells. (B,C) High-power micrograph of im-
pregnated Purkinje cells seen in A. Arrows indicate its axon.

Figure 6 Coronal section (100 µm) obtained using a cryostat through the mouse cerebrum and
brainstem. Note the many Golgi-stained neurons present in the cerebral cortex, hippocampus,
and midbrain.

Figure 7 Low-power micrograph of an area in the (A) mouse striatum showing Golgi-impregnated
medium spiny neurons and (B) mouse cerebral cortex. The sections were counterstained with FD
Cresyl Violet SolutionTM so that the nuclei of nonimpregnated neurons and glia were stained blue.
Note that Golgi-impregnated neurons only represent a fraction of the neuronal population.

temporarily stored in xylene for a few hours
before coverslipping.
Cleaning the laboratory bench after com-
pletion of the protocol typically requires
10 min.
In summary, Golgi-Cox staining with
the FD Rapid GolgiStainTM Kit generally
requires 3 weeks to prepare impregnated sec-
tions for microscopic observations. This in-
cludes 2 weeks for impregnation, 4 days for Du

17 of 19
Current Protocols in Neuroscience
 tissue preparation (including sectioning), and
1 day for staining, depending on the number
of brains to be processed. As described above,
however, hands-on time is only about 5.5 hr for
1 mouse brain or 31 hr for 10 mouse brains.
Acknowledgments
I wish to thank all my colleagues at FD
NeuroTechnologies for their direct and indi-
rect contributions and assistance during the
preparation of this article. I would like to thank
Dr. Chuanjiang Yu for taking excellent pho-
tographs, Tony Hoang for preparing the valu-
able Troubleshooting Guide, and Muyang Liu
for preparing the bibliography.
I am very grateful to Tony Hoang, Thanh
Tran, Muyang Liu, and Connie Du for care-
fully going through the manuscript, provid-
ing thought-provoking comments, and im-
proving writing style and readability of the
text. I would also like to express my sin-
cere appreciation to Chuo Zhao, Kewen Feng,
Yang Yuan, and Jane Wang for preparation
of excellent Golgi-impregnated sections and
staining.
I am particularly indebted to a wonder-
ful team, which includes Dr. Chuanjiang Yu,
Tony Hoang, Thanh Tran, Chuo Zhao, Yang
Yuan, and Jane Wang for making Supporting
Information Video 1 of our Golgi-Cox staining
protocol.
Conflict of Interest
Fu Du is the founder and owner of FD Neu-
roTechnologies, Inc, the company that pro-
duces the FD Rapid GolgiStain Kit used in
this article.
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Internet Resources
http://www.fdneurotech.com
Provides additional information on the FD Rapid
GolgiStainTM Kit and allows for placing an order
online
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