Reprinted from: Brain and Behavior. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.
),
National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:108-111.
THE GOLGI TECHNIQUES FOR STAINING NEURONS
Shankaranarayana Rao BS and Raju TR
For many years the Golgi method has been
recognized as one of the most elegant procedures
for studying the morphology of neurons.
Successful impregnation of brain tissue with this
method provides a complete picture of neuronal
morphology. Its uniqueness lies in the fact that
only a small fraction of the neuronal elements
are stained; thus, it is possible to obtain thick
brain sections in which cell processes can be
traced for long distances through surrounding
unstained tissue. But, as it is universally
recognized, the Golgi method is capricious and
most often, desperately unpredictable. Groups of
nerve cells and fiber tracts are stained randomly,
and many times selected parts appear completely
stained while neighboring areas are completely
devoid of staining.
In recent years the Golgi methods have taken
second place to other techniques involving
intracellular labeling, yet these new methods never
came close to matching the overview of entire brain
areas that good Golgi preparations can provide.
The fact remains that even after 130 years of its
discovery, the technique is increasingly used, not
only in its primary role in qualitative histology, but
as a keystone in the new quantitative neurobiology,
experimental neurology, neuropathology and as
the essential hand maiden of the electron
microscopy. The Golgi method continues to provide
a view of the nervous system both unique and
indispensable.
The main strength of the Golgi method lies in
its capacity to reveal all components of the
nervous system; neurons, glia and vascular
system. Only a small percentage of the neurons
in any one area (1-10%) are impregnated in a
single preparation. Of the neurons rendered
visible, virtually all portions can be seen. The cell
body, dendrites, dendritic substructures (spines)
and at least part of the axon are visible. It provides
a panoramic view of the entire neural element in
black on a yellow or pale orange background.
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The basic method was initially discovered by
Camillo Golgi (1873), which involved the exposure
of brain tissue to dichromates followed by
impregnation with heavy metal ions either silver or
mercury. The original method of Golgi was very time
consuming i.e., immersion of the tissue in potassium
dichromate for several months and subsequent
impregnation in dilute silver nitrate for many days.
The genius of Santiago Ramon Y Cajal was
compounded by profound biological insight, skillful
exploitation of existing techniques and invention
of new ones, consummate artistry and an
enormous capacity for sustained hard work. In
1887, he took up a neurohistological staining
method, which, although introduced by Golgi, was
modified and referred to as ‘rapid Golgi staining
procedure’. Cox (1891) developed another
modification called the Golgi-Cox method that is
particularly useful for tracing dendritic
arborization. Hortega del Rio (1928) used formalin
with the dichromate salt and chloral hydrate and
this variation of rapid Golgi method is particularly
useful for studying the neuroglia, small granule cells
and the cerebellum (Scheibel and Scheibel, 1978).
Fox et al. (1951) described a variation of rapid Golgi
method for use with formalin fixed brain tissue. This
method is useful for the adult tissue. Many other
variations of the above mentioned methods have been
described in the literature. A modified rapid Golgi
method for different ages was developed in our
laboratory (Gundappa and Desiraju, 1988,
Shankaranarayana Rao et al. 1993, 2001a,b). The
common artifacts in the Golgi technique are precipitation
of flat orange chromate crystals and some large brown
or black masses (Scheibel and Scheibel, 1978).
The Golgi methods comprise several different
metallic impregnation techniques. These methods
have in common, the property of impregnating
cytoplasm with metallic salts, which render the
entire profile of an individual cell and its processes
visible in histological preparation suitable for light
or electron microscopy.
General principles of the Golgi methods
The original techniques of Golgi and all the
modifications that have been subsequently
proposed can be grouped into two main categories
depending on processing duration and type of
metallic salts used.
a. Those leading to the deposit of silver chromate
are usually referred to as “Rapid Golgi
method”. The precipitate is an unstable
“lipoprotein-chromic silver complex” limited
by the cell membranes.
b. Those producing a deposit of metallic mercury
and probably complex oxides of mercury are
commonly named “Golgi-Cox methods”.
Both variants of Golgi technique share four
essential features that differentiate them from
other metallic stains;
i. Only a few cells are stained out of large
number of cells in the tissue.
ii. In the few cells that take the stain, the
metallic deposit is massive and invades the
soma or perikaryon as well as the ramifying
processes that originate from cell body.
iii. The remaining unstained structures
compose a transparent background
against which the stained cells stand out
very clearly and distinctly.
iv. There are no staining gradations; the
structures are either completely opaque to
light or perfectly transparent.
The selective staining of only a few neurons in
the CNS has been difficult to understand when the
Golgi technique is employed. Some investigators
are of the opinion that the selective staining may
be due to differences in physiological levels of
activity within the cell that is resting, highly active
or fatigued, which make them susceptible to the
applied stains. Accordingly, the pH and metabolic
state of the neural tissue at the time of fixation and
interaction of heavy metal salts have a major role
to play in staining.
1. Rapid Golgi Staining
The working method standardized and adopted
in our laboratory (Gundappa and Desiraju, 1988,
109
Shankaranarayana Rao et al. 1993, 2001a, b) is
described below;
Composition of rapid-Golgi fixative
Potassium dichromate - 5g
Chloral hydrate - 5g
Glutaraldehyde - 8 ml
Formaldehyde - 6 ml
Dimethyl sulfoxide - 10 drops
All these chemicals are dissolved in 100 ml of
distilled water.
Impregnation solution (0.75% Silver nitrate):
750mg of silver nitrate (AgNO3) dissolved in 100ml
distilled water.
Tissue Processing: Rats are deeply anesthetized
and decapitated quickly. The brain is exposed by
an incision along the midline of the skull. A small
amount of fixative is poured on the exposed brain
immediately. The brain regions of interest (for e.g.
hippocampus / cortex) are dissected out. Tissues
are transferred to 25ml of the fixative in an amber
colored bottle. The tissue is processed as described
below;
1st day: 60 ml of the fixative is prepared and
about 25 ml of the fixative is poured into an amber
colored bottle and the tissue blocks are immersed
in the fixative. The bottle is closed tightly with a
lid and kept in the dark chamber. The remaining
35 ml of the fixative is stored in the refrigerator.
2nd day: The fixative in which the tissue is
immersed in the previous day is gently poured out.
The tissues are rinsed by adding a small amount of
fixative, the remaining fixative is poured into the
bottle and kept in the dark chamber.
3rd day : 40 ml of fixative is freshly prepared.
The fixative from the bottle is poured out and tissue
blocks are rinsed once or twice with the fresh
fixative. About 25ml of the fixative is poured into
the bottle and kept in the dark.
4th day: The tissue blocks remained undisturbed.
5th day: The tissue blocks are rinsed several times
in 0.75% aqueous solution of silver nitrate
(AgNO3) till the reddish brown color of the
potassium dichromate-silver complex disappeared.
Tissues are then placed in about 25 ml of 0.75%
AgNO3 solution and kept in the dark for a
minimum of 48 hours.
Tissue embedding : After 48 hours, the tissue
pieces are placed in a petridish and the silver
deposits were brushed off gently. The tissue is
blotted and then dehydrated in absolute alcohol
for 10 minutes. After dehydration, the tissue
blocks are carefully mounted on required plane
of cutting onto block holders and shell embedded
with paraffin wax. After the wax was set, the block
holder is mounted onto a sliding/ sledge
microtome.
Sectioning : 120mm thick sections are taken
in sledge or sliding microtome. The sections are
collected in a petridish containing distilled alcohol.
Clearing and mounting: The sections are lifted
gently with a brush. The sections are blotted dry
and then transferred to xylene, in a petridish for
clearing. Clearing was com-plete, once the sections
started sinking and also when the sections became
translucent. The sections are then mounted serially
on slides with DPX and cover slipped. The slides
are air dried for a week. The dendritic arborization is
analysed using Sholl’s method (Shankaranarayana
Rao et al. 1993, 2001a, b) and also by computerized
image analysis (Vyas et al., 2002)
2. Golgi-Cox method
Although they are slow, the Golgi-Cox method
is most reliable of the Golgi techniques, especially
for demonstrating the dendritic architecture and
axonal arbors of neurons in the mammalian CNS.
The whole brain of a rat or mouse may be immersed
in the Golgi-Cox fixative. A detailed account of
modified Golgi-Cox protocol which was
standardized and adopted in our laboratory
(Ramon-Moliner, 1970; Hayashi et al., 2004; Saura
et al., 2004) has been described below.
Golgi-Cox fixative
The following stock solutions are prepared.
1. Potassium dichromate, 5% solution in distilled
water (dissolved in warm distilled water).
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2. Mercuric chloride, 5% solution in distilled water
(dissolved in hot distilled water).
3. Potassium chromate, 5% solution in distilled
water (dissolved in cold distilled water).
a. Mix 5 volumes of potassium dichromate
solution and 5 volumes of mercuric chloride
solution; separately mix 4 volumes of
potassium chromate solution and 10
volumes of distilled water.
b. Slowly pour the first mixture into the
diluted potassium chromate solution while
stirring continuously. A dark reddish/
yellowish deposit (mercuric chloride) will
form. The fluid must be free of this deposit
before brains are placed in it. Wait approx.
2 hrs until no more deposit forms.
c. Filter the fluid through a retentive filter (e.g.
Whatman #42). This solution is now ready
for use.
Preparing brains
1. Put brains (as fresh as possible; no perfusion or
fixation) on glass wool or gauge in clean bottles.
2. Cover the brains with the Golgi-Cox solution
and leave at room temperature and in the dark.
3. After 2 days, change the Golgi-Cox solution.
4. Expose tissue to the solution for 6-10 weeks.
Dehydration : After 6-10 weeks of
impregnation in the Golgi-Cox fixative, the brains
are processed further in the following order;
50% ethanol : 1 hour
70% ethanol : 1 hour
95% ethanol : 2 hour
100% ethanol : 1 hour
Absolute ethanol + Ether (1:1) : 1 hour
Transfer to 15% LVN (Nitrocellulose in ether +
absolute alcohol(1:1)) : overnight
30% LVN : 24 hour
Place in mold harden in chloroform vapors : 48
hours
Store blocks in liquid chloroform
 Sectioning : Cut 120 mm sections in microtome
Processing of sections: Collect sections in 70%
ethanol ! H2O (5 min) ! 5% Sodium carbonate
(20 min) ! H2O (5 min) ! 70% ethanol (10 min)
! 95% ethanol (3 min) ! Terpineol or cedar
wood oil (1 hour) ! Xylene (2 min) ! mount
sections with DPX or canada balsam on a slide.
Adventages of Golgi-Cox method
1. Extreme clarity and sharpness of impregnated
neurons
2. Minimal background precipitation
3. Best results in all aged animals
References
1. Fox, C.A., Ubeda Purkiss, M., Ihrig, H.K. and Biagioli,
D. (1951) Zinc chromate modification of the Golgi
technique. Stain Technology 26 : 109-114.
2. Golgi,C. (1873) Sulla structtura della sostanza grigia
dela ervello. Gazetta Medica Lomberda 33 : 244-246.
3. Gundappa, G and Desiraju, T. (1988) Deviations in
brain development of F2 generation of caloric
undernutrition and scope of their prevention by
rehabilitation : Alterations in dendritic spine
production and pruning of pyramidal neurons of
lower laminae of motor cortex and visual cortex,
Brain Res., 456:205-223.
4. Hayashi ML, Choi S-Y, Shankaranarayana Rao BS,
Jung H-Y, Lee H-K, Zhang D, Chattarji S, Kirkwood A
and Tonegawa S (2004) Altered Cortical Spine
Morphology and Impaired Memory Consolidation in
Forebrain-Specific Dominant Negative p21-activated
Kinase (PAK) Transgenic Mice. Neuron 42(5): 773-787.
5. Hortega del Rio P. (1928) Contribullcional
conocimiento citologieo de los tumores del nervio y
111
quiasna optio. Memorios Sociedad Espanola de
Historia Natural 4 : 7-11.
6. Kiernan JA (1999) Histological and histochemical
methods-Theory and practice, 3rd edition, Butterworth-
Heinemann, Oxford, UK. Pp.385-389.
7. Ramon-Moliner, E (1970) Contemporary research
methods in neuroanatomy. Berlin, Heidelberg, New
York: Springer.
8. Saura CA, Choi S-Y, Beglopoulos V, Malkani S, Zhang
D, Shankaranarayana Rao BS, Chattarji S, Kelleher III
RJ, Kandel ER, Duff K, Kirkwood A Shen J (2004) Loss
of Presenilin Function Causes Impairments of Memory
and Synaptic Plasticity Followed by Age-Dependent
Neurodegeneration. Neuron 42(1):23-36.
9. Scheibel, M.E. and Scheibel, A.B.(1978) The methods
of Golgi. In: Neuroanatomical research techniques,
Robertson, (ed.), Vol.2, Academic press, New York,
pp.90-114.
10. Shankaranarayana Rao BS, Desiraju T and Raju TR
(1993) Neuronal plasticity induced by selfstimulation
rewarding experience in rats a study on alterations
in dendritic branching in pyramidal neurons of
hippocampus and motor cortex. Brain Res. 627 (2) :
216224.
11. Shankaranarayana Rao BS, Govindaiah, Laxmi TR,
Meti BL and Raju TR (2001) Subicular lesions cause
dendritic atrophy in CA1 and CA3 pyramidal
neurons of the rat hippocampus. Neuroscience 102(2)
: 319-327.
12. Shankaranarayana Rao BS, Madhavi R, Sunanda and
Raju TR (2001) Complete reversal of dendritic atrophy
in CA3 neurons of the hippocampus by rehabilitation
in restraint stressed rats. Curr. Sci. 80(5) : 653-659.
13. Vyas A, Mitra R, Shankaranarayana Rao BS and
Chattarji S (2002) Chronic Stress induces contrasting
effects on dendritic structure of hippocampal and
amygdaloid neurons. J. Neurosci., 22(15) : 6810-6818.