Histological Methods
Chapter 1: Histology & it’s methods of study (p. 1-16)
Chapter 2: The Cytoplasm (p. 49-50) table 2-6
Versatest: Methods in Histology
There are many principles of preparation of tissues for study. (The Friendly Doctor Can
Identify Every Sick Specimen).
➢ Tissue Collection: tissue biopsies are collected.
➢ Fixation: Whenever a piece of tissue is collected, it will undergo fixation. This
serves two important goals; preservation and fixing the tissue. Tissues and cell
contain lysosomes; this is an organelle in the cell which contains many enzymes
that break down tissue. If the cell dies, those enzymes are released and will start
breaking down degrading cells. Preservation prevents this degradation.
Fixing the tissue prevents the tissue from changing shape and volume. The tissue
that will be processed will optimally resemble the way it was when it was
collected.
Fixation for light microscopy is usually done using Formalin (formaldehyde in
water). Electron microscopy is usually done by Glutaraldehyde,
which is better at preserving the fine subcellular structures.
The way these preservatives work, is by opening up the double
bond that links the Oxygen, and forming bridges. They can then crosslink amino
acids in a protein chain, to inactivate (denature) the protein. They can also do
this in living tissue, which makes preservatives hazardous.
➢ Dehydration: For the preparation of histological slides, the objective is to cut
very thin (micrometres thin) slices of tissue. In order to do that, the tissue must
be embedded in a firm substance, as the tissue itself is too soft. The firm
substance we use is mostly paraffin (wax) for light microscopy. For electron
microscopy (and sometimes also light microscopy) plastic resin can also be
used. Wax is not soluble in water, but it is soluble in xylene; thus water in the
tissue will first be replace with xylene. Xylene is soluble in alcohol. The tissue is
dehydrated by passing it through graded washes of ethanol, which replaces all
the water in the cell. Xylene is solvable in ethanol, which then allows for the wax
to penetrate the cells.
➢ Clearing: For microscopy, it helps if the cell is clear. In this step the ethanol is
replaced with an organic solvent. This gives the tissue a transparent appearance.
This organic solvent is often the same compounds as used in the last step of the
dehydration; xylene.
➢ Infiltration: In this step, the tissue is placed in the wax (liquid paraffin) in order to
achieve the firmness needed for sectioning. The tissue is infiltrated with the
liquid paraffin. This also evaporates the clearing solvent.
➢ Embedding: An important step is to orient the tissue in the desired plane, when
placing it in the paraffin mold. Once the wax has infiltrated, it is allowed to
harden.
➢ Sectioning: A microtome is usually used for this step; the paraffin block with the
tissue embedded is mounted on the microtome, which creates sections that are
typically 5-12 µm thick (LM) or 50-100 nm (EM). The sections are then placed on
glass slides (LM) or metal grids (EM). An average human cell is 10 µm.
, A cryostat can also be used, which is in essence a microtome placed in a freezer.
The tissue sample is then quickly frozen in liquid nitrogen. This freezing allows for
the firmness needed for the sections. This is a quicker procedure (e.g. during
surgery), and also useful is dissolvable compounds must be retained (e.g.
membrane compounds or lipids, that tend to dissolve during the dehydration). It
is however more difficult to obtain very thin sections.
➢ Staining: Most cells (and thus tissues) and extracellular matrixes are colourless.
Stains make structures visible. Dyes (stains) depend on the chemical
composition of the tissue. This can be based on many things, like acidity, charge,
hydrophobicity, or a combination. But they most often behave like acidophilic or
basophilic compounds forming linkages.
Basic dyes react with basophilic compounds (thus compounds that are acidic
themselves) (e.g. DNA, RNA, GAGs). Examples are Haematoxylin, Toluidin blue,
Alcian blue, etc.
Acidic dyes react with acidophilic compounds (e.g. Mitochondria, most proteins,
collagens, granules). Examples are Eosin, Orange G, acid fuchsin.
These dyes are often combined; staining allows for visualization of structures,
but also gives a clue to the chemical properties of the tissues.
The most universally used histological stain is called the H&E stain,
which combines the Haematoxylin and Eosin stains.
The Haematoxylin is a basic dye, which reacts with acidic structures,
and will stain structures containing DNA and RNA, such as the
nucleus and the ribosomes. Eosin will stain the proteins and fibres.
Another frequently used stain is the Periodic Acid-
Schiff (PAS) stain. This is a stain with a compound
that specifically reacts with carbohydrates, more
specifically sugars and sugar-derivatives. Thus it
stains hexose (carbohydrates, e.g.
polysaccharides) like glycogen, glycoprotein,
proteoglycans, glycolipids.
Cells that produce mucus (goblet cells; present in
e.g. intestinal tract) stain
poorly with H&E staining, but they stand out with the PAS-stain.
Other stains include the Trichrome (Masson) stain, which stains
collagen blue, muscle red and elastic fibres yellow/brown.
Connective tissue can secrete three types of fibres; elastic fibres,
reticular fibres and collagen fibres. The Aldehyde Fuchsin (Weigert)
stain, specifically stains Elastic fibres.
The Silver (Golgi) stain stains reticular fibres,
but also neuronal cells.
,There are all general stains. But there are also more specific stains, discussed below.
Enzyme histochemistry; this is used to localize specific enzymatic activities in cells
and tissues, e.g. phosphates, dehydrogenases, peroxidases, and requires unfixed or
mildly fixed tissue; the alcohol, paraffin and hot temperatures can inactivate enzymes.
The principle is that the tissue is immersed in a solution with substrate (the substrate
depends on whichever enzyme we’re interested in); the enzyme is then allowed to act
on the substrate. The product then precipitates, which is visible identifiable.
Immunohistochemistry (IHC) combines immunology and histology. It’s a way of
demonstrating the presence of a specific protein, antigen or molecule against which an
antibody has been formed. It’s a highly sensitive, antibody-based method to detect
specific proteins.
An antigen here is a foreign substance which induces an immune response and causes
the production of antibodies. Antibodies are a serum protein produced in response to,
and counteracting, a specific antigen.
In situ hybridization (ISH) is another way of demonstrating the presence of certain
molecules in tissues or cells. Conceptually it is very similar to IHC, but it does not use
antibodies. Instead, it uses a complementary single strand RNA/DNA molecule.
It is specific annealing of a labelled probe to complementary sequences of a target
nucleic acid (RNA/DNA). Then, histological methods are used to detect and visualize
this.
Microscopy
There are many types of microscopy. Light microscopy (LM) has a magnification up to
1000X; it can show individual cells and has many possibilities;
• Brightfield microscopy,
• Phase-contrast microscopy,
• Polarizing microscopy,
• Fluorescence microscopy,
• Confocal microscopy.
Electron microscopy (EM) has a magnification up to 10.000x or higher. It can show
subcellular structures.
• Scanning EM,
• Transmission EM.
, Histological Artifacts
Histological artifacts are structures or properties that are not normally present in living
tissue. In some situations, the presence of an artifact can compromise an accurate
interpretation (e.g. diagnosis). See slides.
Chapter 1: Histology & it’s methods of study (p. 1-16)
Chapter 2: The Cytoplasm (p. 49-50) table 2-6
Versatest: Methods in Histology
There are many principles of preparation of tissues for study. (The Friendly Doctor Can
Identify Every Sick Specimen).
➢ Tissue Collection: tissue biopsies are collected.
➢ Fixation: Whenever a piece of tissue is collected, it will undergo fixation. This
serves two important goals; preservation and fixing the tissue. Tissues and cell
contain lysosomes; this is an organelle in the cell which contains many enzymes
that break down tissue. If the cell dies, those enzymes are released and will start
breaking down degrading cells. Preservation prevents this degradation.
Fixing the tissue prevents the tissue from changing shape and volume. The tissue
that will be processed will optimally resemble the way it was when it was
collected.
Fixation for light microscopy is usually done using Formalin (formaldehyde in
water). Electron microscopy is usually done by Glutaraldehyde,
which is better at preserving the fine subcellular structures.
The way these preservatives work, is by opening up the double
bond that links the Oxygen, and forming bridges. They can then crosslink amino
acids in a protein chain, to inactivate (denature) the protein. They can also do
this in living tissue, which makes preservatives hazardous.
➢ Dehydration: For the preparation of histological slides, the objective is to cut
very thin (micrometres thin) slices of tissue. In order to do that, the tissue must
be embedded in a firm substance, as the tissue itself is too soft. The firm
substance we use is mostly paraffin (wax) for light microscopy. For electron
microscopy (and sometimes also light microscopy) plastic resin can also be
used. Wax is not soluble in water, but it is soluble in xylene; thus water in the
tissue will first be replace with xylene. Xylene is soluble in alcohol. The tissue is
dehydrated by passing it through graded washes of ethanol, which replaces all
the water in the cell. Xylene is solvable in ethanol, which then allows for the wax
to penetrate the cells.
➢ Clearing: For microscopy, it helps if the cell is clear. In this step the ethanol is
replaced with an organic solvent. This gives the tissue a transparent appearance.
This organic solvent is often the same compounds as used in the last step of the
dehydration; xylene.
➢ Infiltration: In this step, the tissue is placed in the wax (liquid paraffin) in order to
achieve the firmness needed for sectioning. The tissue is infiltrated with the
liquid paraffin. This also evaporates the clearing solvent.
➢ Embedding: An important step is to orient the tissue in the desired plane, when
placing it in the paraffin mold. Once the wax has infiltrated, it is allowed to
harden.
➢ Sectioning: A microtome is usually used for this step; the paraffin block with the
tissue embedded is mounted on the microtome, which creates sections that are
typically 5-12 µm thick (LM) or 50-100 nm (EM). The sections are then placed on
glass slides (LM) or metal grids (EM). An average human cell is 10 µm.
, A cryostat can also be used, which is in essence a microtome placed in a freezer.
The tissue sample is then quickly frozen in liquid nitrogen. This freezing allows for
the firmness needed for the sections. This is a quicker procedure (e.g. during
surgery), and also useful is dissolvable compounds must be retained (e.g.
membrane compounds or lipids, that tend to dissolve during the dehydration). It
is however more difficult to obtain very thin sections.
➢ Staining: Most cells (and thus tissues) and extracellular matrixes are colourless.
Stains make structures visible. Dyes (stains) depend on the chemical
composition of the tissue. This can be based on many things, like acidity, charge,
hydrophobicity, or a combination. But they most often behave like acidophilic or
basophilic compounds forming linkages.
Basic dyes react with basophilic compounds (thus compounds that are acidic
themselves) (e.g. DNA, RNA, GAGs). Examples are Haematoxylin, Toluidin blue,
Alcian blue, etc.
Acidic dyes react with acidophilic compounds (e.g. Mitochondria, most proteins,
collagens, granules). Examples are Eosin, Orange G, acid fuchsin.
These dyes are often combined; staining allows for visualization of structures,
but also gives a clue to the chemical properties of the tissues.
The most universally used histological stain is called the H&E stain,
which combines the Haematoxylin and Eosin stains.
The Haematoxylin is a basic dye, which reacts with acidic structures,
and will stain structures containing DNA and RNA, such as the
nucleus and the ribosomes. Eosin will stain the proteins and fibres.
Another frequently used stain is the Periodic Acid-
Schiff (PAS) stain. This is a stain with a compound
that specifically reacts with carbohydrates, more
specifically sugars and sugar-derivatives. Thus it
stains hexose (carbohydrates, e.g.
polysaccharides) like glycogen, glycoprotein,
proteoglycans, glycolipids.
Cells that produce mucus (goblet cells; present in
e.g. intestinal tract) stain
poorly with H&E staining, but they stand out with the PAS-stain.
Other stains include the Trichrome (Masson) stain, which stains
collagen blue, muscle red and elastic fibres yellow/brown.
Connective tissue can secrete three types of fibres; elastic fibres,
reticular fibres and collagen fibres. The Aldehyde Fuchsin (Weigert)
stain, specifically stains Elastic fibres.
The Silver (Golgi) stain stains reticular fibres,
but also neuronal cells.
,There are all general stains. But there are also more specific stains, discussed below.
Enzyme histochemistry; this is used to localize specific enzymatic activities in cells
and tissues, e.g. phosphates, dehydrogenases, peroxidases, and requires unfixed or
mildly fixed tissue; the alcohol, paraffin and hot temperatures can inactivate enzymes.
The principle is that the tissue is immersed in a solution with substrate (the substrate
depends on whichever enzyme we’re interested in); the enzyme is then allowed to act
on the substrate. The product then precipitates, which is visible identifiable.
Immunohistochemistry (IHC) combines immunology and histology. It’s a way of
demonstrating the presence of a specific protein, antigen or molecule against which an
antibody has been formed. It’s a highly sensitive, antibody-based method to detect
specific proteins.
An antigen here is a foreign substance which induces an immune response and causes
the production of antibodies. Antibodies are a serum protein produced in response to,
and counteracting, a specific antigen.
In situ hybridization (ISH) is another way of demonstrating the presence of certain
molecules in tissues or cells. Conceptually it is very similar to IHC, but it does not use
antibodies. Instead, it uses a complementary single strand RNA/DNA molecule.
It is specific annealing of a labelled probe to complementary sequences of a target
nucleic acid (RNA/DNA). Then, histological methods are used to detect and visualize
this.
Microscopy
There are many types of microscopy. Light microscopy (LM) has a magnification up to
1000X; it can show individual cells and has many possibilities;
• Brightfield microscopy,
• Phase-contrast microscopy,
• Polarizing microscopy,
• Fluorescence microscopy,
• Confocal microscopy.
Electron microscopy (EM) has a magnification up to 10.000x or higher. It can show
subcellular structures.
• Scanning EM,
• Transmission EM.
, Histological Artifacts
Histological artifacts are structures or properties that are not normally present in living
tissue. In some situations, the presence of an artifact can compromise an accurate
interpretation (e.g. diagnosis). See slides.
