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. 2004:104-107.
THE NEUROHISTOLOGICAL TECHNIQUES
Shankaranarayana Rao BS, Titus ADJ and Raju TR
Histology is the study of cells and tissues. To
observe the tissue sections and to visualize details
at light microscopic level, it is necessary to impart
colour to the cellular elements to be studied. The
neurohistology is important to neurobiological
experiments to find the exact location of
manipulations i.e., electrical recordings, lesions,
brain damage, etc. Different parts of a nerve cell
can be stained with special stains e.g. Cresyl violet-
stains the Nissl bodies, Luxol fast blue- stains
myelin, Golgi stains all the neuronal compartments
like dendrites, spines, axon and cell body.
Following steps are undertaken to achieve
staining in the histology.
1. Fixation of the tissue
2. Processing of tissue
3. Sectioning
4. Mounting of sections
5. Staining
TISSUE FIXATION
Histological studies require stabilization of cells
by fixation with minimal alterations from living
state and virtually no loss of tissue constituents.
Tissues are composed of soluble and insoluble
substances. Hence, cellular constituents not bound
to solid structures can easily lead to diffusion
artifacts. Fixation is the process, which prevents
the tissue decomposition (autolysis), helps in
hardening the tissue to preserve the
cytoarchitecture of the biological structure. If
fixation is not proper many artifacts like autolysis
or edema of tissue can occur.
Fixation of the Nervous Tissue
Choosing the best mode of fixation is
undoubtedly as crucial as identifying the best
fixatives for a particular staining procedure. The
two modes of fixation that are suitable for nervous
tissue are either “vascular perfusion” or
“immersion fixation”. The former is the better
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option because of the following reasons: (i) Fixation
begins immediately after cessation of the systemic
circulation, (ii) Even fixation of heterogeneous
tissue components is possible, and (iii) Fixation in
vivo enables the cells to retain their original shape
and structure.
The tissue is fixed either by chemical or physical
method.
a. Chemical Method of Fixation
For most purposes, tissue fixation is done by
chemical reactions. A good commonly used fixative
for light microscopic studies is 10% buffered
formalin volume/volume (pH 7.0).
b. Physical Method of Fixation
Physical methods of fixation include freeze
substitution, freeze-drying, freeze etching and inert
dehydration. These methods are, however, more
tedious to perform and are commonly associated
with ice crystallization artifacts. They are,
therefore, employed for specific purposes only.
Vascular Perfusion
For conducting vascular perfusion, one can
devise a simple unit or make use of automatic
perfusion pump. An important factor to be taken
into consideration is the perfusion pressure, which
should remain constant. Fluctuation in the pressure
during perfusion can introduce artifacts.
One of the simplest perfusion units is shown
below in the diagram (Figure 1). Perfusion pressure
is obtained by holding the fixative containers above
the level of animal. The height of the container with
respect to the animal determines the applied
pressure. The containers are usually held about
120-150cm above the animal for intra-arterial
perfusion whereas for intravenous perfusion, much
lower height is (20-30cm) required.
Requirements For Perfusion
1. 10% formalin
2. 9% saline
3. Surgical instruments PROCESSING OF THE TISSUE
4. 19 gauge needles The fixed tissue has to be processed further to
5. Sodium pentabarbitone get good sections. Tissues are either freezed or
embedded in paraffin wax or hard material like
Perfusion Method celloidin, low viscocity nitrocellulose, araldite,
epoxy resin, etc for sectioning. The paraffin wax is
The animal is weighed and the amount of
commonly used for processing the brain tissue.
fixative required is 2-3 times the weight of the Accordingly, the procedure adopted in our
animal. The animal is deeply anesthetized with laboratory is described below.
sodium pentabarbitone (50 mg/kg b.w.). The
thorax was cut opened and heart was exposed; a
needle connected to the tubing from the fixative Tissue Processing in Paraffin Wax
bottle, is inserted into left ventricle. The right In this method the tissue is hardened and
atrium was cut open to drain out the blood and embedded in the paraffin wax. This makes it easy
fixative (see Figure 1). First 20-30ml of saline is to retain sections. The progressive replacement of
passed transcardially to flush out the blood, then water by processing tissue in different grades of
perfused with formalin. After perfusion the animal alcohol (dehydration), and cleared in xylene/
is decapitated, the brain is shelled out and kept in chloroform.
10% formalin for minimum 2-3 days for proper
fixation. Commonly used fixatives are 10% Procedure
formalin, 4% paraformaldehyde, 1% Para
The tissue is kept under running water to remove
formaldehyde + 1.25 glutarldehyde, etc.
formalin. The tissue blocks are processed for 24
hours as follows.
1. Wash the fixed tissue in water for 1hr
2. 70% alcohol/ overnight
3. 90% alcohol/3hrs
4. Absolute alcohol-1 /1hr
5. Absolute alcohol-2 /1hr
6. Absolute alcohol-3 /1hr
7. Chloroform-1 - overnight
8. Chloroform: 2 - 3hrs (till the tissue appear
clear & translucent)
9. Paraffin wax -1 (M.P 52ºC) : 1-2hrs
10. Paraffin wax -2 (M.P 52ºC) : 1-2hrs
11. Paraffin wax -3 (M.P 52ºC) : 1-2hrs
12. Paraffin wax - 4 (M.P 52ºC) : overnight

Figure 1. Fixation of an anaesthetized rat by perfusion from
the left ventricle to the right atrium. The saline is run through
first, until the effluent fluid is clear. The fixative (usually a
formaldehyde or glutaraldehyde solution) is then perfused until
the whole body of the animal is hard and inflexible. About 100-
200 ml of fixative required for 200-300g rat, and the whole
procedure takes 15minutes (Thorball and Tranum-Jensen, 1983).
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Embedding
L- Shaped metal pieces are used to form paraffin
blocks. Wax is poured in the block and air bubbles
are removed. The tissue block is quickly placed with
the help of a warm forceps and oriented as desired.
The block is allowed to cool and the block is
trimmed and a wooden block is fitted to it and kept
ready for sectioning.
Sectioning of paraffin blocks
The paraffin block sections are obtained
using rotary microtome. The microtome is
adjusted for suitable thickness of 5 to 10µm. The
level of knife and block is appropriately adjusted
and sections were taken. The sections are
collected in water. The sections are mounted on
albumen or gelatine coated slides and the slides
are kept on the slide warmer. The paraffin will
melt slowly and it helps in spreading of the
sections and also the sections will adhere to the
surface of the slide.
Mounting the sections
1. Sections are taken on egg albumin coated slides.
This gets coagulated and once water evaporates
sections adhere firmly to the slide.
2. The slides are dipped in xylene (tissues
impregnated with paraffin are nearly
impermeable to stains hence xylene is used to
remove paraffin.
3. The sections are processed using series of
alcohol grades (Downgrading- 100%, 90%,
80%, 70%).
4. Sections treated with the stains as described
below.
5. Ascending grades of alcohol (70%, 80%, 90%,
100%) – (to remove all water before they can
be treated with clearing agent, as water is
immiscible with the clearing agent (xylene)
6. Xylene is used for clearing.
7. Mounting – once the sections are mounted it is
encased in a medium of suitable refractory
index for microscopic observations and also
allowed it to be preserved.
STAINING OF BRAIN SECTIONS
Various stains have been developed which
have particular affinity to certain parts of the
cell elements. These stains render color to these
elements so that microscopic study is easier on
the contrasting background. The commonly
used stains in our laboratory are described
below.
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1. HAEMATOXYLIN AND EOSIN STAINING
Haematoxylin stains the nuclei blue and eosin
stains the cytoplasm in shades of blue. This method
is used for identification of tumors, recognition of
inflammatory cells and certain inclusion bodies.
Solutions required
Haematoxylin powder : 6gm
Absolute alcohol : 300ml
Distilled water : 300ml
Glycerin : 300ml
Glacial acetic acid : 300ml
Potassium alum in excess
Ripen in sunlight for several weeks
For differentiation use acid alcohol: 1%
hydrochloric acid in 70% alcohol
Eosin: 0.5% eosin in distilled water.
Staining Procedure
1. The slides are dipped in distilled water
2. Stain in haematoxylin /10-15 min
3. Wash the slides in running water till the section
is blue
4. Differentiate in 1% alcohol till the nuclei
becomes blue
5. Wash the slides in running water / 5min
6. Stain with Eosin/ 1-2min
7. Rinse in distilled water followed by tap water
8. Dehydration done through a series of alcohol
grades (70%,80%,90% &absolute)
9. Clearing in xylene
10. Mounted in DPX
2. CRESYL VIOLET (NISSL) STAINING
First used by Nissl in1892. Cresyl violet stains the
Nissl substance. Nissl stains are basic stains as they
are able to stain the Nissl bodies, which contains
acidic ribonucleo protein. The method adopted in
our laboratory to evaluate the cell density and
degeneration are described in detail (Govindaiah et
al. 1997; Latha Devi et al 2003; Kiran et al 1998;
Ravikumar et al. 1998). Cresyl violet stainsthe Nissl
substance as purple with colorless background.
Staining solution Staining Procedure
0.1% aqueous cresyl violet with 15 drops of 1. The slides are dipped in 70% alcohol
glacial acetic acid 2. Stain with LFB - 2hrs @ 60ºC
3. Wash in 95% alcohol followed by distilled
Differentiator water
95% alcohol : 100ml 4. Differentiate in lithium carbonate for10sec. and
Chloroform : 2ml 70% alcohol alternately under microscopic
Acetic acid : 8 drops or 70% alcohol control till only nerve fibers are stained
5. Counterstaining is done with 1% Cresyl violet
Staining Procedure stain - 10 min.

1. The slides containg brain sections are dipped 6. Slides are washed in running water
in chloroform -1 min 7. Up grading is done through though a series of
2. 100% alcohol - 2 min alcohols 70%, 80%, 90% and absolute alcohol

3. 90% alcohol - 2 min 8. Cleared with xylene : 1-2 min

4. 80% alcohol - 2 min 9. Mounted in DPX and cover slipped

5. 70% alcohol - 2 min

6. Distilled water - 10min
7. 0.1% Cresyl violet at 60ºC for 30-45 min
8. Allowed to cool and left in distilled water for10 min
9. 70% alcohol - 2 min
10. 80% alcohol - 2 min
11. 90% alcohol - 2 min
12. 100% alcohol - 2 min
13. Xylene for clearing : 2-3 dips
14. Mounting in DPX and cover slipped
3. LUXOL FAST BLUE (LFB) STAINING
Luxol fast blue is used to stain myelin sheath,
hence for the demonstration of myelinated nerve
fibers and tracts. Myelin stains blue, nuclei stains
purple and Nissl substance stains purple
(Munirathinam et al. 1997).
Staining solution
1% Luxol fast blue in absolute alcohol
1gm in 1 liter or 0.1gm +100ml 95% alcohol and
10% acetic acid added to each 100ml to make the
solution stable.
Differentiator
0.05% Lithium carbonate
References
1. Govindaiah, Shankaranarayana Rao BS, Raju TR
and Meti BL (1997) Loss of hippocampal CA1 neurons
and learning impairment in subicular lesioned rats.
Brain Res. 745 (1-2) : 121126.
2. Kiran B, Shankaranarayana Rao BS, Raju TR and
Bindu PN (1998) Spinal cord ischaemia induced
excitotoxicity and neurodegeneration : Attenuation
by () deprenyl and magnesium sulfate. Med. Sci. Res.
(London) 26 (2) : 8992.
3. Latha Devi, Diwakar L, Raju TR, Bindu M. Kutty,
Spatial learning impairment and selective
neurodegeneration of hippocampal and
entorhinocortical neurons in ventral subicular
lesioned rats. Brain Res. 2003; 960: 9-15.
4. Munirathinam, S., Rao, M.S., Ramamohan, Y. and
Raju, T.R. (1997) Regeneration of the olfactory tract
following neonatal lesion in rats. Exp. Neurol. 144:
174-182.
5. Ravikumar R, Lakshmana MK, Shankaranarayana
Rao BS, Meti BL, Bindu PN and Raju TR (1998) ()
Deprenyl attenuates spinal motor neuron
degeneration and associated locomotor deficits in
rats subjected to spinal cord ischemia. Exp. Neurol.
149 (1) : 123129.
6. Thorball, N. and Tranum-Jensen, J. (1983) Vascular
reactions to perfusion fixation. J. microscopy 129:
123-139

70% Alcohol
0.1% Cresyl violet stain- as a counter stain
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