Methods, Specific Techniques and Stains in Microscopy
What is Cytochemistry?
In cell biology and biology in general,
cytochemistry is the study of the (bio-chemical) chemical constituents of the
cell. Using cytochemistry, it has also become possible for scientists and
technicians to localize various chemical components or elements, which may be
enzymatic or non-enzymatic in nature.
A number of techniques
are used for the purposes of localizing these chemical components (of both the
cells and organelles) as well as their transformation in relation to the
functioning of the cells of interest.
This technique is used for the purposes of
detecting a ligand bond to a given target site or cell. For this technique,
molecules with a high affinity for each other are used. As such, it have proved
to be an important technique for detecting neuropeptides (group of compounds
that act as neurotransmitters - these are typically short-chain polypeptides).
In affinity cytochemistry, one of the most common methods is the streptavidin
system (Biotin-Avidin system). Avidin (the egg white derived glycoprotein) has
a high affinity for Biotin.
The Biotin molecules can be attached/ coupled to a
protein, which would allow the protein (biotinylated protein) to be able to
bind to more than one molecule of avidin. This makes it possible to visualize
any proteins that may be present in low amounts. That is, the ability to
visualize proteins that are in low amounts is enhanced.
This method can also be
used together with microscopy. Here, avidin is covalently linked with electron microscope
markers allowing the Biotin-Avidin system to be used for the purposes of
studying a wide range of biological structures and processes.
Couple biotin molecules on
to the protein to have a biotinylated protein
Cells of interest are then
exposed to the biotinylated protein or ligand - Here, it is important to perform
biotinylation under gentle (suitable conditions) conditions so as to preserve
the biological activities of the protein
This is then cooled or
fixed at low temperatures depending on the system being used
Lastly, the biotinylated
ligand or protein is detected by either of the following:
Avidin linked to gold ferritin
Cytochemistry Immunoperoxidase Technique
This is a staining technique that is based on enzymatic
detection of antigen - antibody complex using a biotin labeled secondary antibody
and subsequent signal amplification using the biotin-peroxidase and strepavidin
In this method, the visualization of the antigen is made possible by
the reaction between the peroxidase and diaminobenzine which forms a brown
precipitate at the site of the PR protein (pathogenesis-related).
In Situ Hybridization
In situ hybridization is a technique that is
used for the localization and detecting of specific mRNA sequences in tissues
that have been morphologically preserved or in cell preparations. This is
achieved by the hybridization of the complementary strand of a nucleotide probe
to the sequence of interest.
This technique works using the same principle as
normal hybridization process. In this case, there is isolation of DNA/RNA,
separating it in gel, blotting it on to the nitrocellulose and finally probing
it with a complementary sequence. With in situ hybridization, the only
exception is that a probe is used for the purposes of detecting the specific
nucleotide sequences within the cells or tissue of interest.
This being a sensitive technique, the threshold
levels of detection range between 20 and 50 copies of mRNA per cell.
This method has often been used to indicate the
localization of gene expression in their respective environments. Here, the
labeled RNA or a DNA probe is used to hybridize a given target mRNA/DNA
sequence within the sample of interest. It becomes possible for the RNA
or DNA probe to be detected using an antibody that can detect the label of the
One of the problems that is likely to be
experienced with this technique is with regards to the sequence to be detected. Here
the sequence tends to be at a lower concentration. It may also be masked
because of associated protein or protected within the cell/cellular structure.
In this case, probing the tissue/cell of interest requires that one increases
permeability of the cell as well as the visibility of the nucleotide sequence
so as to probe while at the same time avoiding destroying the structure of the
Some of the probes used in this technique include:
Oligonucleotide probes - They are synthetically
produced through an automated chemical synthesis process. They have a number of
advantages including being resistant to RNases in addition to the fact that
they are small (40 to 50 base-pairs) which makes it easier during penetration
into the cell or tissue of interest. Lastly, they are single stranded which is
a big advantage in that the possibility of renaturation is greatly minimized.
Single stranded DNA probes - Despite being larger
(200 to 500 bp) these probes have advantages similar to Oligonucleotide probes.
Double stranded DNA probes - This probe is produced
by including the sequence of interest in bacteria. This is then replicated and
lysed with the DNA being extracted and purified. Lastly, the sequence of
interest is excised with a restriction enzyme.
RNA probes - These probes have been shown to be very
thermostable and resistant to digestion by RNases which are some of its
Also referred to as immunogold staining, this is a technique that is commonly used in electron microscopy as a staining
technique. As such, it is often used for identification, localization as well
as the distribution of proteins, antigens and a number of other macromolecules.
Although it is more similar to the conventional immunofluorescence techniques
used in microscopy, this technique provides an added dimension compared to the
fluorescence assays given that it makes it possible to clearly observe the
localization and distribution of macromolecules at ultrastructural level and at
high magnifications and high resolution.
This technique is largely used for detecting
proteins, antigens as well as various other macromolecules. In particular, it
is used for detecting these molecules when they are located on the surface of
the cell or any other biological specimen.
It has also proved to
be an important technique for other specimen that may be ultra-thin sectioned.
Here, it is largely used to examine the exterior and interior of such specimen.
Post-Embedding Labeling is the technique that is
only used to detect proteins, antigens and various other macromolecules located
in the interior regions of the specimen of interest.
Negative Stain Immunogold Labeling Technique - This technique is used
for the purposes of detecting proteins, antigens or other macromolecules that
may be on the surface or the interior of biological specimen.
Immunogold labeling is a powerful technique,
making it one of the few or only techniques that can be used to probe cells
untrastructurally. This is largely due to the fact that the process is devised
to attach gold probes to secondary antibodies and consequently to primary
antibody that help reveal the presence of antigen.
Some of the advantages of immunogold techniques
It is relatively easy to
It is particularly suitable
for real-time monitoring of the ultrastructure and antigens.
It can help improve
assessment of target markets on cellular microenvironment.
It provides good
intratechnique comparison and reassessment as well as crucial information for
Cytochemical stains are mostly used in
hematopathology for such purposes as:
To characterize blast cells
Confirm the diagnosis of
hairy cell leukemia
To differentiate between
leulaemoid reactions and leukemia
For classifying leukemia
abnormalities and enzyme deficiencies in myeloid disorder
Basically, the process involves a reaction
between the enzyme being used and the substrate to produce a given color. With
regards to cytochemical stains, there are both enzymatic and non-enzymatic
Sudan black B
Periodic acid Schiff
This is an enzyme that is located in the
granules of both the myeloid and monocytic cells. As a stain, it is positive in
cells of granulocyte series. Its activity increases with the maturation of
myeloid cells while the immature myeloid cells tend to have very little to no
This stain works by splitting the hydrogen peroxide
in the presence of chromogenic electoron donor, which results in the formation
of an insoluble product.
This enzyme (e.g. alpha naphthyl acetate
esterase) is mostly found in monocytic cells in large amounts. In myeloid and lymphoid
cells, it is present in little amounts. It is typically used for the purposes
of identifying monocytes. As a result, lymphocytes will often stain focally
while the myeloid cells stain very weakly. This type of stain is largely used
for the purposes of diagnosing acute myelogenous leukemia with monocytic
The Periodic Acid Schiff (PAS)
This stain is mostly used for the purposes of
demonstrating glycogen and related mucopolysaccharides. When used for Myeloid
or monocytic blasts, the outcome is often weakly positive or negative. However,
it is positive in erythroid population in erythroblastic leukemias.
Sudan Black B
This is a lipophilic dye that is typically used
for the purposes of staining intracellular phospholipids and other lipids. It
functions by irreversibly binding to an unidentified granule in granulocytes or
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