Techniques/Methods, Staining and Tests
Histochemistry is an important technique that is
used for the visualization of biological structures. As such, it is concerned
with the identification and distribution of various chemical components of
tissues through the use of stains, indicators as well as microscopy.
Essentially, identification and distribution of chemical constituents of
tissues is achieved through the exploitation of unique chemical environments in
cells, heterologous expression techniques as well as enzymatic activities.
This method is particularly important for the
detection of ion levels (ferric ions). Because it can help
detect the presence of ferric ions, this technique is used to determine the level
of these ions in such organs as the spleen and bone marrow. It can be
used to tell whether there are excessive amounts of the ion as observed in
hemochromatosis (excessive levels of ferric ions with deposits in the liver and
pancreas) or hemosiderosis where deposits can be found in the liver, spleen and
the lymph nodes.
In plants, this technique has also been shown to help
understand the homeostasis of ferric ions.
With this technique, ferric ions present in the
tissue will combine with ferrocyanide resulting in the formation of a pigment
called Prussian blue (ferric ferrocyanide). In plants, the technique is also
based on the conversion of ferrocyanide into insoluble crystals (Prussian
blue) in the presence of Ferric ions under acidic conditions. Prussian blue
(resulting from the reaction) is bright blue in color, which indicates the
presence of ferric ions.
In Perl's reaction technique, a known positive
control tissue is used as control while 10 percent formalin is used as a
- Microwave oven
- Acid cleaned glassware
- Non-metallic forceps
- 5 percent potassium
- 5 percent hydrochloric acid
- Sample section (cut to
* It is important to use gloves, goggles and a
lab coat because some of the chemicals used for this procedure can cause
- Deparaffinize the section(s) and hydrate with distilled water
- Microwave for about 30
seconds and allow the specimen to stand in the working solution for about 5
minutes in the fume hood. The section can be treated with
the working solution (acid ferrocyanide) for between 10 and 30 minutes.
- Rinse/wash the section
using distilled water
- Stain (lightly) the section
with 0.5% aqueous neutral red or 0.1% nuclear fast red. This part of the
procedure is used to stain the nuclei.
- Rapidly wash the section
using distilled water
- Dehydrate the section,
clear and mount on the microscope stage for viewing
When viewed under the microscope, blue parts are
indicative of iron while the red and pink parts indicate the nuclei and
Von Kossa Technique
This is also an ion based technique that is used
in histochemistry. It is a more sensitive technique that can be used to
identify the presence of calcium deposits on cyst fluids, ductal ectasia and
papillomatosis. However, excessive amount of calcium may be found in any given
part of the body and can be demonstrated using the Von Kossa technique.
Although this technique is used to demonstrate the presence of calcium, it
demonstrates an anion rather than the calcium ion itself.
For this technique, the sample section is
treated with the solution of silver nitrate and is reduced and the calcium (if
present in the sample) is reduced by the strong light and replaced with
deposits of silver. As a result, it is visualized as metallic silver.
Requirements for Von Kossa technique
- A control sample (a tissue
that contains known calcium deposits or undecalcified bone)
- 10 percent formalin
- Glassware (clean)
- 60 watt lamp
- A mirror or a foil
- 5 percent of silver nitrate
- 5 percent hypo (sodium
- Deparaffinize and hydrate
the section using distilled water
- Place the section in the
silver solution (in a glass jar) and place it in bright light (or in front of
the 60 watt lamp). Place a mirror or a paper foil behind the jar so as to
reflect the light. Leave it standing for about one hour or until the calcium
- Rinse the section in
- Stain using 5 percent hypo
solution for about 5 minutes
- Wash the section using tap
water or rinse in distilled water
- Introduce the sample to
nuclear-fast Red for about 5 minutes
- Wash using distilled water
- Dehydrate and mount for
A black color indicates the present of calcium
(calcium salts), red indicates the nuclei while the cytoplasm will appear pink.
This technique is dependent on dyes that are
soluble in lipids. Some of the most common dyes used include:
- Sudan VI
- Sudan black
- Oil Red O
- Nile blue
Lipid staining is a useful technique that is
used for demonstrating intracellular lipids in various tissue sections.
For this technique, the dye is more soluble in
the lipid, which allows it to be more demonstrated than in the vehicular
solvent. The dyes used in this technique are all interchangeable, which means
that they can be substituted for each other for the staining process.
- ORO (Oil Red O) solution
- Glycerine jelly mounting
- Cut the sample to obtain
sections of between 8 and 10 microns and air dry
- Rinse the section with 60
- Stain the section with the
Oil Red O working solution for about 15 minutes
- Rinse the specimen with 60 percent
- Dip the section in Alum
hematoxylin a few times in order to stain the nuclei
- Rinse in distilled water
- Mount the specimen in water
or in glycerin jelly
Red color indicates the lipid while blue
coloration indicates the nuclei.
Lipid staining technique is useful for showing
the normal distribution of lipids as well as disease-related lipid
Protein and Amino Acids
Some of the methods used for specific amino acids include:
- Millon's reaction
- Sakaguchi reaction
- Tetrazotized benzidine reaction
Millon's reagent is used for detecting amino
In this technique, the mercurous and mercuric nitrate
(components of the reagent) reacts with hydroxybenzene radicals to form a
compound that is red in color. Tyrosine contains the phenolic group, which
forms the red coloration in the presence of Millon's reagent. The compound
formed through this reaction is called mercuric fumarate.
Milton's method procedure:
- Add about 2ml of the
protein solution into a test-tube
- Add a few drops of the
reagent (Millon's reagent) in to the test-tube that contains the original
- Using a tube holder, hold
the test-tube over boiling water or flame and boil for about half a minute.
If the solution turns reddish in color after
boiling, then tyrosine is present in the solution.
The Sakaguchi reaction test involves the use of the Sakaguchi reagent. This reagent is composed of
1-naphthol and sodium hypobromite and forms a reddish compound when mixed with
the sample containing a
The test is positive for any amino acid that
contains the guanidine group in Arginine. Therefore, the Guanidine group in an
amino acid will react with the α-Naphthol and alkaline hypobromite in the
reagent to give of a red-colored complex indicating the presence of such
- Add 1 ml of the protein
solution in to a test-tube
- Add 2 or 3 drops of 40
percent of sodium hydroxide, 2 drops of ethanolic a-Naphthol and 5 drops of
- Mix the contents in the
- If a red-color complex
forms when shaking the contents, then it is positive that the solution contains
Arginine or a protein containing Arginine.
Tetrazotized Benzidine Reaction
Although it has been shown to be less effective,
Tetrazotized benzidine is used in histochemistry to detect non-collagen
proteins. The procedure involves the coupling of Tetrazotized benzidine with
beta naphthol or Hyaluronic acid.
The method, commonly referred to as Tetrazotized benzidine helps in the detection of such non-collagen proteins as
tyrosine, histidine as well as tryptophan.
Nucleic Acids and DNA
This is a relatively new technique that is used
for demonstrating DNA in tissue sections. It is a sensitive means of detecting
aldehydes, which makes it the ideal method for detecting the presence of DNA.
Here, the section is treated with dilute hydrochloric acid in order to remove
The sugar part that remains reacts as an aldehyde ultimately forming
a visible color. Therefore, this method can be said to be divided in to two
1/ The first part of the procedure is the
hydrolysis phase that involves the use of 5N HCl, ambient temperature for 40
minutes. This step is aimed at separately selecting 2 purine bases (adenine and
guanine) which are removed from the DNA molecule.
2/ The second step is the
staining phase. The reagent used is preferred because it is highly
selective for DNA rather than RNA. Here, RNA does not react because of the
presence of hydroxyl on carbon 2 of ribose, which prevents the acid (HCl) from hydrolyzing
sugar. The reaction is also precise for the localization of DNA given that
deoxyribose radicals are bound to phosphoric acid of the apurinic acid molecule
following the removal of purine bases.
Some of the stains used for both DNA and RNA
- Methyl green pyronin stain
PAS reaction (Periodic Acid Schiff)
This is one of the most popular histochemical techniques
for the detection of glycogen. It has been shown to be one of the best
techniques for demonstrating carbohydrates in tissue. In this technique, the
periodic acid oxidizes tissue carbohydrates to produce aldehyde groups. This group
then condenses with the reagent to form a bright red coloration to demonstrate
the tissue component with carbohydrate attachments.
The diastase and a-amylase in the reagent act on
the glycogen and depolymerize it into smaller sugar units (maltose and
glucose) which are then washed out of the section.
- Deparaffinize and hydrate
the slide using distilled water
- Place the section in
preheated diastate solution (at 37 degrees centigrade) for about an hour
- Wash the sample in running
water for about 5 minutes
- Place the sections in 0.5
percent periodic acid solution for about 5 minutes
- Wash the section in
- Place the section in Schiff
reagent for about 15 minutes
- Wash the section for about
a minute in 0.55 percent potassium metabisulfite in order to remove excess
- wash in running tap water
for about 10 minutes
- Counterstain using Harris's
hematoxylin with acetic acid for half a minute
- Wash with running water
- Dehydrate with two changes
using absolute alcohol, clear with xylene and mount to view
Some of the other stains used for staining
Whereas histochemistry includes a number of
techniques used for the visualization of various chemical components in
tissues, immunochemistry involves the study of identities and functions of
components of the immune system (particularly antibodies).
immunochemical methods are based on the selective, reversible and non-covalent
binding of antigens by antibodies. This allows for these methods to be used to
quantify antigens or antibodies.
All the immunochemical methods/techniques
depend on a highly specific and sensitive reaction between antigens and
antibodies. There are a number of immunochemistry techniques based on the type
of reaction, reagents and samples that are used. These include:
Particle methods - This is the technique
where the antigen-antibody interaction is observed. It includes a number of
methods such as Immunoprecipitation, Immunoelectrophoresis, Immunofixation.
Label methods - With label methods, either the antigen or the
antibody is labeled allowing for the antigen-antibody reaction to be observed.
Immunoassay and competitive binding are examples of label methods. Some of the
other methods include:
- Immunoelectron Microscopy
Return to Immunofluorescence - In Microscopy, Applications, Direct and Indirect Methods
Return to Immunohistochemistry - Microscopy, Techniques, Cancer Research
Return from Histochemistry to Microscope Research and Information
Boguslaw Samotus, Maria Leja, Andrzej Scigalski,
Jerzy Dulinski, Robert Siwanowicz (1982) Determination of tyrosine by a
modified Millon's reaction and its application to potato tuber extracts.
Culling C.F.A., (1974) Handbook of histopathological
and histochemical techniques Ed. 3
Butterworth, London, UK.
Dandekar (1 January 2004). Practicals And Viva
In Medical Biochemistry. Elsevier India. p. 28. ISBN 978-81-8147-025-6.
Kiernan. J.A., (1999) Histological and
histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.
Luna, Lee G (1960) Manual of Histologic Staining
Methods of the Armed Forces Institute of Pathology (Third Edition). American
Registry of Pathology.
Lee Bergma, Stephanie Bechtel and Stefan Wiemann
(2006) Immunochemical Methods, Localization.
Symonds DA (1990) Use of the von Kossa stain in identifying occult
calcifications in breast biopsies.
Suzuki M, Shinohara Y, Fujimoto T (2013)
Histochemical detection of lipid droplets in cultured cells.