Interpretation of histological sections: Stains used in histology

Van Gieson

The van Gieson stain is a very common stain used to highlight the difference between collagen and other connective tissue such as muscle tissues. It is often used to identify the characteristic arrangement of fibers in different types of tumours. The stain uses a mixture of picric acid and acid fuchsin to penetrate the tissue sample, causing collagen to become red. Surrounding muscle tissues and blood cells are stained yellow.

Picric acid molecules are very small, and therefore penetrate all types of tissues. However, they are only fully retained in red blood cells and muscle, as these have a fine, close texture. Acid fuchsin has larger molecules, which penetrate collagen, displacing the picric acid molecules. This causes collagen to be stained differently from muscle and red blood cells, allowing differentiation. Nowadays, a substance called ponceau S is often used as a substitute for acid fuchsin, as it is slightly more efficient.

To conduct the van Gieson stain, the following steps are carried out:

• The section is rehydrated and then cleared using xylene
• The section is stained with celestin blue-hematoxylin sequence to colour the nuclei  
• Excess is rinsed off using water
• The section is immersed in Van Gieson’s stain for 1-5 minutes
• It is rinsed again with distilled water
• It is then dehydrated with ethanol and cleared using xylene
• The section is mounted using a resinous medium

Toluidine blue

Sometimes referred to as tolonium chloride, toluidine blue is a type of metachromatic dye, that is acidophilic, meaning that it stains acidic tissues. It is particularly attracted to nucleic acids, and is therefore used to stain tissues with high concentrations of DNA and RNA. When in contact with Toluidine Blue, nucleic acids become blue in colour, whilst mucins and cartilage become purple. It is used to identify mast cells granules, mucins, and cartilage.

The basic procedure to carry out a Toluidine Blue stain is as follows:

• The tissue section is hydrated using distilled water
• The section is immersed in Toluidine Blue for a few minutes
• Excess is washed off with distilled water
• It is then dehydrated with ethanol and cleared using xylene
• The section is mounted using a resinous medium

Alcian blue

Alcian blue is one of the alcian dyes introduced way back in 1948. The other alcian dyes include alcian yellow and alcian green. This is a family of polyvalent basic dyes contains 2-4 isothiouronium groups having positive charge.

Alcian blue is a phthalocyanine dye which shows specificity for substances such as glycosaminoglycans and acid mucins. To characterize the subtypes of acid mucins, the pH of the alcian blue solution is varied and adjusted. The alcian blue stain causes acid mucins and mucosubstances to appear blue, and nuclei to appear reddish pink when counterstain neutral red is used. Alcian blue dyes are water soluble, and appear blue as they contain copper. They attach to sulfate and carboxylated acid mucopolysaccharides and glycoproteins, and dye binding is purely electrostatic. This staining is performed to see the mucoid degeneration and to identify acid mucins which are released by various connective and epithelial tissue tumors. The procedure of staining is usually carried out as follows:

• The section is hydrated with distilled water
• It is submerged in alcian blue (time in the alcian blue can vary)
• It is then washed in tap water, and rinsed in distilled water
• Nuclear-fast red is added as a counterstain for 5 minutes
• It is then dehydrated with ethanol and cleared using xylene
• The section is mounted using a resinous medium

Giemsa

This is a blood stain that can be used histopathologically. It was designed primarily to demonstrate malarial parasites. It was later used in histology because of its high quality staining capabilities of chromatin and nuclear membranes. It stains human and pathogenic cells differently, therefore, it is used in the diagnosis of many diseases as it stains human cells purple, and bacterial cells pink, so that they may be differentiated. It is also used to stain blood cells, so that their composition and structure may be observed.

Nuclei are stained purple, and cytoplasm is stained blue to pale pink, depending on cell type. The stain differentiates the granules of different blood cells by staining them different colours. Basophils show dark blue granules and eosinophils show orange granules.

The staining process is as follows:

• A film of blood is air dried
• It is then immersed in Giemsa stain for 1 to 2 minutes
• It is submerged in deionised water for 2 to 4 minutes, and then rinsed in deionised water
• It is then air dried

Reticulin

Reticulin staining employs the use of silver impregnation of a section to highlight reticulin fibers (type III collagen). It is mainly used in histopathology of the liver, but can also be used to assess abnormalities in the spleen, bone marrow and kidneys. In the liver, both necrosis and cirrhosis cause irregular patterns of reticulin. Changes in reticulin can also signal the presence of tumours.

The stain causes the fibers to be stained black, which contrasts with a paler grey or pink background. During the staining procedure, the tissue must first be oxidised and then sensitized with iron alum before silver is added. Once silver has been added, it must be reduced using formalin so that it becomes visible. The nuclei can also be counterstained red using nuclear-fast red, to make them visible.

The process of the stain is outlined below:

• The section is hydrated using distilled water
• It is immersed in potassium manganate solution for 5 minutes
• It is washed in tap water
• 5% oxalic acid is added, and then washed off with distilled water
• The section is sensitised using iron alum for 10 minutes
• It is washed in tap water and rinsed in distilled water
• It is then dipped in silver solution a number of times, followed by distilled water
• It is immersed in 10% formaldehyde solution for 30 seconds
• It is washed in distilled water
• Gold chloride is added for 1 minute and rinsed away with distilled water
• The section is then immersed in 5% hypo for 1 minute and washed off in tap water
• Nuclear-fast red is added as a counterstain for 5 minutes
• It is then dehydrated with ethanol and cleared using xylene
• The section is mounted using a resinous medium

Nissl

Nissl staining is used to visualise Nissl substance (clumps of rough endoplasmic reticulum and free polyribosomes), which is found in neurons. This stain distinguishes neurons from glia and the cytoarchitecture of neurons can be well studied with the help of this stain. A loss of Nissl substance can signify abnormalities such as cell injury or degeneration, which in turn can indicate disease.

A commonly used dye in this stain is called Cresyl Echt Violet Acetate, which is mixed in a solution with distilled water. This stains Nissl substance a dark blue or dark purple colour.

The following steps are used to carry out a Nissl stain:

• The section is hydrated with distilled water
• It is immersed in Cresyl Echt violet acetate for 2 minutes
• It is washed in distilled water
• It is then dehydrated with ethanol and cleared using xylene
• The section is then mounted using a resinous medium

Orcein

The orcein stain is used to identify the inclusion bodies of viruses. These are viral particles within human cells and are visible using light microscopy, unlike viruses themselves. The Orcein stain is commonly used to diagnose hepatitis B, which causes inclusion body formation in hepatocytes. Orcein is composed of mixture of amino- and hydroxyphenoxazone compounds. The result of the stain is that inclusion bodies are stained a dark brown-purple colour. Proteins that are associated with copper also become stain dark purple. The procedure uses a solution of Orcein combined with 70% ethanol, and hydrochloric acid.

The process of conducting an Orcein stain is as follows:

• The section is hydrated using distilled water
• Dip in potassium permanganate solution for 10 minutes
• Wash in water
• Dip in 5 % oxalic acid until colourless
• Wash in tap water
• 0.5% Periodic acid is then added for 5 minutes
• It is then washed in tap water and rinsed in distilled water
• The section is then immersed in Orcein solution between 4 and 16 hours at room temperature
• It is rinsed in ethanol
• It is then dehydrated with ethanol and cleared using xylene
• The section is mounted using a resinous medium

Sudan black B

It is a non ionic, hydrophobic dye used to identify lipids and lipofuscins. Lipofuscins are age pigments occurring in older people in permanent cells like neurons and heart cells. Lipofuscin results from a build of lysosomes that have absorbed indigestible parts of cells. As its name suggests, Sudan Black B stains lipofuscins black. It is commonly used to stain lipids and fats, hence the fact it stains lipofuscins is important. It can also stain red blood cells black as well.

To conduct a Sudan Black B stain, the following steps are involved:

• The sample is hydrated using alcohol
• It is then immersed in sudan black overnight
• It is then rinsed in ethanol, and washed in tap water
• The section is then mounted using an aqueous mounting medium

Masson’s trichrome

Trichrome stains, as the name indicates, are mixture of three dyes used to differentiate the muscles, collagen fibers, fibrin and erythrocytes in connective tissue. One of the three dyes is usually nuclear stain and the other two dyes mainly differentiate collagen and muscle fibers.

Masson’s trichrome is one of the commonly used trichrome stains used to highlight the difference between collagen and muscle tissues like van Gieson. It is widely used to assess the collagen in different pathologies, like in liver cirrhosis or tumours. Three different dyes in this stain have different sized molecules, which penetrate tissues differently. Where larger molecules can penetrate, smaller ones are displaced. First, an acidic dye such as Biebrich scarlet is used, followed by phosphotungstic and phosphomolybdic acid, and finally a fibre stain such as Light Green. Weigert's iron hematoxylin is also used, but as a fixative at the start of the procedure. After the stain has been carried out, nuclei appear blackish or blue, muscle and fibrin appear red, and collagen appears green.

The usual staining steps for paraffin sections are listed below:

• The section is rehydrated and then cleared using xylene
• It is then cleaned with alcohol and rinsed in distilled water
• It is  stained with Weigert’s iron haematoxylin for 5 to 10 minutes
• The section is rinsed again with distilled water
• It is then immersed in Biebrich scarlet acid fuchsin for 5 to 10 minutes
• Excess is rinsed off with distilled water
• It is then stained with phosphotungstic and phosphomolybdic acid for 10 minutes
• Immediately after it is stained with light green for 5 minutes
• Excess is again rinsed off with distilled water
• The section is then dehydrated with ethanol and cleared using xylene
• It is mounted using a resinous medium

Mallory’s trichrome

This stain also differentiates between collagen and muscle fibres. Among the three dyes, the first one is diluted acid fuchsin, the second is diluted phosphomolybdic acid and the third is a mixture of orange G, methyl blue, oxalic acid and distilled water. At the end of the procedure, nuclei and muscle cells appear red, collagen appears blue, and erythrocytes become orange. This stain is quite common to detect changes in liver and kidney histopathological samples. The Mallory’s trichrome staining involves the following steps on paraffin embedded sections:

• The section is brought to water using xylene and ethanol
• It is immersed in acid fuchsin for 2 minutes
• It is then rinsed with distilled water
• It is then stained with phosphomolybdic acid for 2 minutes
• It is rinsed again with distilled water
• It is then immersed in the orange G solution for 15 minutes
• It is rinsed with distilled water
• It is then dehydrated with ethanol and cleared using xylene
• The section is mounted using a resinous medium

Azan trichrome

The Azan trichrome stain, sometimes referred to as the Heidenhain’s Azan Trichrome stain is also used to stain muscle and collagen. Therefore, it can be used to differentiate between muscle and collagen tissue, as well as to identify diseases such as liver disorders. It is a slightly improved version of  Mallory’s trichrome.

It distinguishes cells from extracellular components and stains muscle fibres red, cartilage and bone matrix blue. Like the Mallory phosphomolybdic acid orange G and aniline blue solutions are used, however instead of using acid fuchsin to stain nuclei, it uses a dye called azocarmine, which is combined with acetic acid and distilled water. Aniline blue is used as a counterstain to azocarmine to dye the nuclei. The procedure results in red nuclei, orange muscle cells, and blue collagen, allowing them to be differentiated under the microscope. The staining procedure is as follows:

• The section is brought to water with xylene and ethanol
• It is then immersed in the azocarmine solution for an hour, at 50 degrees Celsius
• It is then rinsed using distilled water
• The section is then placed in a solution of aniline and 100 mL ethanol, combined with 0.1 mL hydrochloric acid
• Rinse with distilled water
• It is then immersed in the phosphomolybdic acid for 2 hours
• It is then placed in a mixture of orange G, aniline blue, acetic acid and distilled water for between 2 and 3 hours
• It is again rinsed in distilled water
• It is then dehydrated with ethanol and cleared using xylene
• The section is mounted using a resinous medium

Cason’s trichrome

Like the other trichrome stains, this stain is also used to differentiate collagen. Therefore its applications involve the diagnosis of disorders to do with collagen abnormalities. It stains nuclei and cytoplasm red, collagen blue and erythrocytes orange. The stain used is a mixture of dyes, comprising orange G, acid fuchsin, aniline blue, phosphotungstic acid and distilled water. The process involves the following steps:

• The section is brought to water with xylene and ethanol
• It is then immersed in the stain solution for 5 minutes
• It is rinsed with distilled water
• It is then dehydrated with ethanol and cleared using xylene
• The section is mounted using a resinous medium

PAS (Periodic acid Schiff)

This stain colours glycogen, and is therefore used to look at membranes, mucosubstances as well as the presence of fungus. The process of PAS staining usually involves two steps, the first one is the oxidation reaction with periodic acid leading to the formation of aldehydes, the second step is the demonstration of these aldehydes with the help of Schiff’s reagent.

Fuchsin dye in Schiff reagent gives a range of colors from magenta to purple. The staining process uses periodic acid, haematoxylin, and Schiff’s reagent which comprises basic fuchsin and sodium metabisulfite combined with distilled water and hydrochloric acid. The stain cause nuclei to become blue, and glycogen and fungi to become magenta in colour. PAS is useful in a number of diagnostic applications. For example, it can be used to diagnose glycogen storage disease, certain sarcomas and carcinomas, as well as fungal infections. The procedure is as follows:

• The section is hydrated in distilled water
• It is then placed in periodic acid for 5 minutes
• It is rinse in distilled water
• It is then Immersed in Schiff’s Reagent for 15-30 minutes
• Excess is washed off with tap water
• The section is then counterstained with haematoxylin for 1-3 minutes
• It is then washed in tap water and rinsed in distilled water
• The section then dehydrated with ethanol and cleared using xylene
• Finally it is mounted using a resinous medium

Weigert's resorcin fuchsin (Weigert’s elastic)

As its name suggests, this type of stain is used to colour elastic fibers. It causes them to be stained blue-black, nuclei to become light blue-black, collagen to become pink or red, and other tissues to become yellow.

The solution includes basic fuchsin, which produces a complex that attaches to elastic fibers, causing them to become stained. Weigert's Stain solution also comprised of resorcin, ferric chloride, ethanol, distilled water and hydrochloric acid. Haematoxylin and van Gieson stain are also used as counterstains. The process involves the following steps:

• The section is hydrated and cleared with xylene and ethanol
• It is immersed in Weigert’s solution for up to an hour (minimum 20 minutes)
• It is washed with ethanol
• 1% acid alcohol is added to cause differentiation
• Excess is then washed off in water
• Haematoxylin and van Gieson counterstains are then added
• The section is then dehydrated with ethanol and cleared using xylene
• It is is mounted using a resinous medium

Wright and Wright Giemsa stain

The Wright and Wright Giemsa stains are polychromatic stains because they contain eosin and methylene blue. Giemsa stain additionally contains methylene blue azure and intensifies the nuclear features. The eosin Y is then used to stain cell cytoplasm orange. Both of them are used to stain peripheral blood smear and bone marrow smears. They are used to look at cells as well as their morphology, aiding in the diagnosis of infections and blood diseases such as leukemia. Different blood cell types become stain differently allowing them to be differentiated by the observer.

These changes are listed below:

• Neutrophils have purple nuclei, light pink cytoplasm, and reddish-purple granules
• Basophils have dark blue or purple nuclei, and very dark purple granules
• Eosinophils have blue nuclei, light pink cytoplasm, and red granules
• Red blood cells become red or pink
• Platelets appear purple

The process for staining blood with Wright stain is as follows:

• A drop of blood is placed between glass slides and air dried in a thin layer to be stained
• The smear is then placed in Wright stain for 1 to 3 minutes
• A phosphate buffer is added for double this time
• It is then rinsed with water, and dried by blotting

Aldehyde fuchsin

This stain, sometimes known as Gomori's aldehyde fuchsin stain, was originally created to stain elastic fibers. However, it can also be used to stain other tissues, particularly the beta cell granules in the pancreas. It is also highly selective with few other high affinity basophilic sites, like mast cell granules and cartilage matrix.

Elastic tissue fibers become stained a bluish purple colour, as do beta cell granules and sulphated mucins. Aldehyde fuchsin solution contains a mixture of basic fuchsin, 70% ethanol, concentrated hydrochloric acid, and paraldehyde. Aldehyde fuchsin is commonly used in combination with alcian blue. To carry out the aldehyde fuchsin stain, the following process is used:

• A section is brought to water using ethanol and xylene
• It is then oxidised in 1% potassium manganate
• It is then rinsed in tap water
• The potassium manganate is then removed with 1% oxalic acid
• It is then rinsed in tap water and then ethanol
• The section is immersed in aldehyde fuchsin solution in a sealed container for 15 minutes
• It is rinsed again in ethanol, followed by tap water
• It is then dehydrated with ethanol and cleared using xylene
• The section is mounted using a resinous medium

Immunohistochemistry and immunofluorescence

Immunofluorescence is a technique used in immunohistochemistry. Immunohistochemistry is a branch science that involves using the formation of antibody-antigen complexes to selectively look at certain antigens within a tissue section.  

This technique involves labelling antibodies with a fluorescent marker. The antibodies will bind to a specific antigen, at the site of interest, and the fluorescent marker causes a colour change, which can be viewed under the microscope. This technique is useful for identifying abnormal cells, such as cancerous cells. The specific antigens that the antibodies bind to can be found in a number of areas of the cells, including the cytoplasm, nucleus, lipids, proteins and cell membrane.

First, an unlabelled primary antibody specific to the antigen of interest is added, incubated for 60 minutes, and then washed off using phosphate buffered saline (PBS). Then a labelled secondary antibody is added and incubated for around 30 minutes. This attaches to the primary antibodies. The section is then washed with PBS, and mounted to be viewed under the microscope. This method is known as indirect immunofluorescence staining. The process can also be carried out using just a primary antibody that is labelled, this is known as direct immunofluorescence staining.