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Indian Journal of Experimental Biology
Vol. 51, September 2013, pp. 685-693
Quick Golgi method: Modified for high clarity and better neuronal anatomy
Nisha Patro1, Kamendra Kumar
1 & Ishan Patro
1,2,*
1School of Studies in Neuroscience, Jiwaji University, Gwalior India, 474 011 2School of Studies in Zoology, Jiwaji University, Gwalior India, 474 011
Received 3 December 2012; revised 20 June 2013
The Golgi methods have long been used to study the neuronal soma, axons, dendritic arborization and spines. The
major concerns of the Golgi method have been its unpredictable nature (inconsistency of impregnation of the stain), time
consumed, tissue hardening and clear background, resulting in several modifications to improve the cellular visualization. In
the present work we describe a modification of the rapid-Golgi method that takes the benefit of perfusion fixation (with
rapid-Golgi solution) then post-fixation in the same fixative for 36 h followed by 36 h impregnation in aqueous AgNO3
followed by vibratomy. This modification is simpler, faster and inexpensive, provides a consistent staining of neurons with
good resolution of neuronal soma, dendritic arborization as well as spines with much reduced formation of silver chromate
crystals and background in just 3 days.
Keywords: Cerebellum, Cerebral cortex, Dendritic spines, Purkinje neurons, Pyramidal neurons, Rapid-Golgi fixative
Camillo Golgi in 1873 discovered that impregnation
of brain slices with silver chromate solution resulted
in staining of a small population of neurons in their
entirety1. This is known as Golgi method and
continues to be extremely useful and very frequently
used even today for studying the neuronal
architecture. Over the years the method has been
variously modified. The classical Golgi method was
modified by Cajal referred as rapid Golgi staining
method2. Cajal extensively used this method and
demonstrated the previously unimagined neuronal
morphology throughout the nervous system, most of
which continues to be accepted. However, Golgi
method’s importance was best felt when Golgi’s so
called artefacts with no physiological relevant
structures on the dendritic surfaces turned out to be
the dendritic spines in the studies of Cajal. He
identified the real value of Golgi method and was first
to describe spines as small thorns that protruded from
the dendrites of cerebellar Purkinje neurons2,3
. Now
the dendritic spines are known as actual centres of
information processing with the ability to regulate
their own protein synthesis and degradation4,5
.
Dendritic spines are proposed as the primary sites of
synaptic plasticity6, their number, shape and size
changes in response to variations in their extracellular
environment7, environmental changes
8-10 and with
evolution11,12
. Most researchers today rely on a two-
step procedure of Golgi staining, chromation
(potassium chromate and/or potassium dichromate
solution) and silver nitrate impregnation which
subsequently allow the formation of silver chromate
crystals visualized as brownish-black structures13
.
The only concern of the Golgi method has been
its unpredictable nature. This has resulted in several
modifications in terms of variation in solution
composition and pH14-21
, use of single sections for
staining22,23
, use of microwaves13,24-26
, changing
embedding media27,28
, use of vibratome29
, coating of
brain blocks with egg yolk13
, application of vacuum30
,
auto metallographic (AMG) enhancement31
and
variation in temperature of the tissue incubating
media21,24-26,32
. Most modifications aimed at decreasing
the time required for the procedure, reduced
precipitation leading to a clearer background and
uniform impregnation and uptake of the Golgi stain in
the nervous tissue. Golgi staining method is useful for
labelling cell profiles in the central nervous system,
but takes several weeks. The long period of fixation
turns the tissue brittle creating problems in sectioning.
The deeply stained blood vessels appearing as
background interfere in deciphering the neuronal
structures. The present communication reports a
simpler, faster and efficient modification of rapid-
——————
*Correspondent author
Telephone: +91 751 2442789
E-mail: [email protected]
INDIAN J EXP BIOL, SEPTEMBER 2013
686
Golgi staining involving the perfusion of animal with
rapid Golgi fixative and postfixation in the same
fixative leading to a clear background, reduced blood
vessels staining and clear spines and quicker than the
earlier modifications29,33
.
Materials and Methods Wistar rats (250-300 g) used in this study were
raised in the Animal House of School of Studies in
Neuroscience. The animals were housed in chambers
at 25±2 °C room temperature, approximately 50% RH
and 12:12 h L:D conditions. Pelleted standard rat feed
and water was provided ad libitum. The experimental
protocols were pre-approved by the Institutional
Animal Ethics Committee (501/01/a/CPCSEA). A
total of 27 male rats were used in this study.
Rapid-Golgi staining method
Rapid-Golgi fixative solution (RGF) was prepared
as per Patro et al.29
, viz., 5 g potassium dichromate
(Himedia Laboratories, India), 5 g chloral hydrate
(MERCK, India), 8 mL glutaraldehyde (CDH, India),
6 mL formaldehyde (Qualigens, India), 10 drops of
dimethyl–sulphoxide (Qualigens, India) were mixed
and final volume was made up to 100 mL with
distilled water. Three rats were sacrificed by cervical
dislocation followed by decapitation, their brains were
dissected out and the cerebral cortex and cerebellum
were removed. Approximately 1 cm3 tissue blocks
both from cerebral cortex and cerebellar cortex
(through vermis region) were directly immersed in
Golgi fixative and kept in dark amber bottles,
undisturbed, for 2 days followed by a second change
of Golgi fixative for next 2 days. On the 5th day tissue
blocks were rinsed briefly (2-3 times) with 0.75%
aqueous solution of silver nitrate (Qualigens, India)
and kept submerged in the same solution for 2 days in
dark. The tissue blocks were again rinsed with 0.75%
silver nitrate solution (2-3 times) and kept in the same
solution for 3 days in dark. Subsequently, tissue
pieces were washed properly and carefully in 70%
alcohol to remove precipitates of silver and then the
sections were cut using a Leica Automatic Vibratome
(VT1000S Leica Microsystems, Wetzlar, Germany) at
100 µm thickness. The sections were collected in 70%
alcohol, dehydrated in absolute alcohol (Bengal
Chemicals, India), cleared in xylene (Qualigens,
India) and mounted in DPX (Qualigens, India). The
slides were visualized with Leica DM6000
microscope fitted with a digital camera DFC 310 FX
(Leica Microsystems, Wetzlar, Germany).
Quick Golgi staining method (present modifications)
To validate the benefits of the proposed
modifications in the rapid Golgi method, 12 male
Wistar rats were anaesthetized with ether (Qualigens,
India) vapors and brains were perfusion-fixed
(transcardially) with 200 mL of phosphate buffer
saline (PBS) to washout the blood followed by
100 mL of rapid Golgi fixative29
(P-RGF group)
solution at the rate of 20 mL/min. Another group of
12 rats were perfused only with phosphate buffer
saline (P-PBS group). After perfusion with PBS alone
or PBS followed by rapid-Golgi fixative solution
(P-RGF) the brains were dissected out and the
cerebral cortex and cerebellum were removed.
Approximately 1 cm3 tissue blocks from both P-PBS
and P-RGF group rats were post-fixed in RGF and
impregnated with 0.75% aqueous AgNO3 solution in
the following combinations (n=3): 12 h post-fixation
followed by 24 or 36 h AgNO3 impregnation; 24 h
post-fixation followed by 24 or 36 h AgNO3
impregnation; 36 h post-fixation followed by 12, 24
or 36 h AgNO3 impregnation; 48 h post-fixation
followed by 48 h AgNO3 impregnation. On
completion of the respective duration in AgNO3
solution, tissues were washed properly in 70% alcohol
to remove any precipitates and 100 µm thick sections
were cut using a Leica Automatic Vibratome
(VT1000S Leica Microsystems, Wetzlar, Germany).
The slides were visualized using Leica DM6000
microscope equipped with Leica DFC 310 FX digital
camera (Leica Microsystems, Wetzlar, Germany) and
the LAS V4.2 multifocus module that automatically
captured a stack of images at different focus positions
across the depth of the neurons. These images were
then combined into a single image all in-focus
neuronal processes of the stack. The spine density of
220 neurons (110 Purkinje and 110 pyramidal
neurons) was analyzed, in both the staining methods,
using an ocular micrometer scale fitted in the
eye piece of a microscope. For each neuron, 3
measurements were made. The data were analysed
by t-test using SigmaStat version 3.5 for Windows.
Values of P≤0.05 were considered significant.
Results To get better results faster than the earlier modified
Golgi methods, perfusion fixation with the rapid
Golgi fixative solution followed by combinations of
various durations of post-fixation and development
with AgNO3 have been tried in the present study. We
then looked for low background, blood vessels
PATRO et al.: QUICK GOLGI METHOD
687
staining and crystallization with better cellular
details like cell processes, spines, etc. (Table 1).
Combination of 12/24 h and 24/36 h post fixation
with rapid-Golgi fixative solution followed by AgNO3
incubation could not develop much of the details. In
P-PBS group no details could be seen (Fig. 1A and C,
respectively), while in P-RGF group, the interneurons
and Bergmann glial fibers were visible with moderate
impregnation to the central part of the tissue blocks
(Fig. 1B and D, respectively). The combination of
36 h post-fixation following perfusion with rapid-
Golgi fixative solution and 36 h development with
AgNO3 produced wonderful visualization of cellular
details. This combination resulted in minimum
background, least appearance of Golgi stained blood
vessels, complete cellular details (cell soma, axon,
dendrites and spines) of Purkinje neurons and
visibility of interneuron connections with main
projection neurons (stellate, basket and Golgi cells
with Purkinje neurons, Table 1; Fig. 1F) when
compared with corresponding P-PBS group (Table 1;
Fig. 1E). Post fixation for 48 h followed by 48 h
development with AgNO3 also produced similar
results with hardly any further improvement in P-RGF
group (Fig. 1H). However, perfusion with PBS alone
did not provide any such details (Fig. 1G). As
compared to 9 days in rapid Golgi staining procedure,
this modification helps getting better results in 3 days.
It is thus proposed that a combination of perfusion
fixation with rapid-Golgi solution followed by 36 h
post-fixation and 36 h AgNO3 may be used, thus by
the end of the 3rd
day the tissue is ready for sectioning
and visualization. The results thus obtained are
better than the rapid-Golgi method with earlier
modification30
that takes 9 days.
Further, the rapid-Golgi stained brain sections29
were compared with the present Quick Golgi stained
brain sections of 36/36 h combination. Tissue sections
of the present Quick Golgi method presented clearer
details and contrasting cell profiles (Fig. 2B, D and F;
3B, D and F) as compared to the rapid-Golgi method
(Fig. 2A, C and E; 3A, C and E). This difference in
visibility of cell profiles among two different methods
was due to reduced (or even no) staining of the blood
vessels in the proposed Quick Golgi method resulting
in a clear background thus enabling study of the
complete cellular details. The rapid-Golgi stained
blood vessels, prominently visible in the brain
sections of animal perfused with PBS only ruled out
the doubt that blood and its constituents are
responsible for the background. Good camera lucida
tracings were possible with prominent dendritic
spines against a clear background in the proposed
Quick Golgi stained sections (Fig. 2H and 3H). In
Table 1—Qualitative demonstration of rapid Golgi staining in both cerebral cortex and cerebellum in perfusion fixed with rapid-Golgi
fixative solution (P-RGF), perfusion with phosphate buffer saline (P-PBS) only and rapid Golgi method
Impregnation to
central part
Interneurons
Bergmann glial fibers Purkinje cells/spines Pyramidal
cells/spines
Background
Combinations P-RGF P-PBS P-RGF P-PBS P-RGF P-PBS P-RGF P-PBS P-RGF P-PBS P-RGF P-PBS
12 h fixative &
24 h AgNO3
+ - - - + - - - - - - +
12 h fixative &
36 h AgNO3
+ - - - + - - - - - - +
24 h fixative &
24 h AgNO3
++ - + - ++ - - - - - - ++
24 h fixative &
36 h AgNO3
++ - ++ - +++ - - - - - - ++
36 h fixative &
24 h AgNO3
+++ - +++ - +++ + + - - - - ++
36 h fixative &
36h AgNO3
+++ - +++ + +++ ++ +++ + +++ + - ++
36 h fixative &
12 h AgNO3
+++ - +++ - +++ + + - + - - ++
48 h fixative &
48 h AgNO3
+++ - +++ + +++ ++ +++ + +++ + - ++
Rapid-Golgi
method
+++ ++ +++ +++ +++ +++
- = absent; + = few; ++ = moderate; +++ = enormous
INDIAN J EXP BIOL, SEPTEMBER 2013
688
contrast, because of more background, the camera
lucida tracings were difficult and spine details were
not prominently visible in the rapid-Golgi stained
sections (Fig. 2G and 3G).
There was no significant difference in the dendritic
spine densities of both Purkinje (t=1.579, P = 0.116)
and pyramidal neurons (t=1.125, P = 0.262) when
compared between the rapid Golgi and Quick Golgi
Fig. 1—Photomicrographs showing rapid Golgi stained cerebellar sagittal sections of P-PBS and P-RGF group (n=3). [Green arrows
indicate the Golgi stained fibrous astrocytes, blue arrows indicates Purkinje neurons, pink arrows depicts Golgi stained Bergmann glial
fibers. P-RGF: perfusion with rapid-Golgi fixative solution, P-PBS: perfusion with phosphate buffer saline only. Scale bar = 200 µm.]
PATRO et al.: QUICK GOLGI METHOD
689
Fig. 2—Light microscopic images of the Rapid-Golgi (A, C, E) and Quick Golgi stained (B, D, F) cerebellar sections, showing a
contrasting difference in the clarity of information in Quick Golgi stained preparations. [Red arrows represents the Golgi stained blood
vessels. Scale bar =100 µm.]
INDIAN J EXP BIOL, SEPTEMBER 2013
690
Fig. 3—Photomicrographs showing the rapid-Golgi (A, C and E) and Quick Golgi (B, D and F) stained cortical sections. [Red arrows
indicate the Golgi stained blood vessels (BV). Pyramidal neurons with their processes are clearly visible with both the methods, but with
better cellular details in Quick Golgi method due to reduced staining of the blood vessels. Camera Lucida drawings (1000X) of the main
shaft (MS) and primary branch (PB) of apical dendrite showing the better details of dendritic spines with Quick Golgi procedure (H) than
rapid Golgi (G). Scale bar =100 µm.]
PATRO et al.: QUICK GOLGI METHOD
691
methods, suggesting that the present modification
improves in the qualitative visualization of spine
without compromise in quantitative evaluation (Fig. 4).
The proposed Quick Golgi method involves the
following steps:
1 Anesthetize the animal (rat) with ether vapors.
2 Perfuse transcardially with 200 mL of PBS
followed by 100 mL of rapid-Golgi fixative
solution.
3 Dissect out the brain tissue of interest and cut
tissue blocks of 1 cm3 (or smaller) and post-fix in
the rapid-Golgi fixative for 36 h.
4 Rinse briefly with 2-3 changes of 0.75% aqueous
AgNO3, in dark.
5 Immerse tissue blocks in 0.75% aqueous AgNO3
solution for 36 h in dark amber coloured bottle.
6 Remove any precipitate by rinsing with 70%
alcohol and then store in 70% alcohol.
7 Cut vibratome sections of 100 µm thickness and
collect in 70% alcohol.
8 Dehydrate in 90% and absolute alcohol, clear in
xylene and mount in DPX.
Discussion The importance of Cajal’s Golgi method in
visualization and study of dendritic spines has been
well reviewed by Garcia-Lopez3. Rapid-Golgi method
is frequently used to study the morphology of
neurons, glia and dendritic spines in brain sections.
This technique is also widely used and a very useful
tool in neuroanatomy, CNS pathology and in
estimating the functional status of a brain region
under study in terms of neuronal processes and spines
in them8,9,29
. Dendritic spines are the earliest to be
affected in most neurodegenerative disorders like
Alzheimer’s disease (AD), Parkinson’s disease (PD),
schizophrenia and depression34-36
. The changes are
especially related to the number, size, shape and
origin in the neurons. The available software
(computer program) and hardware (microscope and
related software) have made study of spines easy and
allowed detail study of such structures29,37,38
. Even the
biochemist and molecular biologist can benefit from
this method, as it is easy, procedure is simple, and
requires minimal instrumentation.
Cajal in 1887 modified the original Golgi method
introduced in 1873 by Golgi. It is known as rapid-
Golgi method and has been variously modified largely
based on the type of tissue available (live, post-mortal,
preserved, size, species, etc.) and each modification
has led to a different labeling pattern7,33,39
. The
modifications validated and are in use are many but to
list a few: the rapid Golgi procedure, the Golgi-Cox
technique40,41
; the Golgi-Kopsch method42
; the Del
Rio-Hortega method43
and others.
Most of these variations of original Golgi method
are based on the two step procedure that label the cell
profiles; first, the tissue blocks are immersed in the
chromating solution (either potassium chromate
and/or potassium dichromate) and in the next step the
tissue blocks are immersed in silver nitrate that
produces silver chromate crystals, that allows the
labeling of neurons and other cellular inclusions of
the brain. Other variants replace the silver nitrate with
mercuric chloride and so on.
The major issues with the rapid Golgi method
proposed for modifications are: it takes several weeks
to get results due to long fixation time, the tissue
becomes brittle that causes problem in tissue
sectioning32
; stained particles of blood vessels in the
vicinity of stained neurons and glial cells that
interferes in visualizing and capturing images of
stained cells.
In the present modification the issues like time,
elimination of tissue shrinkage, sectioning, staining of
cellular structures like fine branches of neuronal
processes, uniform clear background, reduced staining
of blood vessels, etc. have been addressed. In addition
to an earlier modification including use of a
vibratome for sectioning that needs no embedding, no
expensive cryostats, ease of cutting sections with a
sharp razor blade, no need of cryopreservation and
less tissue damage due to fragmentation29
have been
proposed.
This modification is novel in the sense that for the
first time perfusion of animals with rapid-Golgi
Fig. 4—Spine density of Purkinje and pyramidal neurons in rapid
Golgi method and quick Golgi method
INDIAN J EXP BIOL, SEPTEMBER 2013
692
fixative solution instead of the immersion fixation or
perfusion with PBS alone before immersion fixation
has been tried. This promotes uniform penetration
and thus impregnation of the rapid-Golgi fixative
(chromating solution), much clearer background,
reduced fragments of precipitate and crystals, uniform
impregnation of the chromating solution, better and
homogenous formation of silver chromate thus better
staining of the neurons, interneurons and glial cells.
This also has the advantages of the vibratomy29,41
as
well as better impregnation of the Golgi solution at
the body temperature of 37±1 °C32
and enables to get
preparation ready for study in just 3 days.
The present modification not only have made the
process quicker but also is easy even for the
researchers without an exposure to neuroanatomy,
such as biochemists and molecular biologists, who
can benefit from this simple method that needs no
special infrastructure/instrumentations.
Acknowledgement Financial support from Indian Council of Medical
Research, New Delhi is thankfully acknowledged.
Kamendra Kumar is a Department of Biotechnology
(DBT) Senior Research Fellow. Facilities, developed
through the DBT-Human Resource Development
and Bioinformatics Infrastructural facility from
Department of Biotechnology, used in this study are
thankfully acknowledged.
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