Periodic Acid-Schiff (PAS) Staining
Periodic Acid-Schiff (PAS) staining is a widely used technique in histochemistry and histological studies to detect the presence of carbohydrates and carbohydrate-rich compounds in biological samples. Carbohydrates are essential molecules that play important roles in various biological processes, such as energy storage, cell signaling, cell adhesion, and immune response. However, carbohydrates are often difficult to visualize under a microscope because they are colorless and water-soluble. PAS staining overcomes this challenge by using a chemical reaction that produces a bright pink or magenta color when it interacts with carbohydrates.
PAS staining was first developed by McManus in 1946, who modified an earlier method by Hotchkiss that used periodic acid to oxidize carbohydrates. McManus added Schiff`s reagent, which is a colorless compound that reacts with aldehydes to form a colored product. Periodic acid oxidizes some carbohydrates to form aldehydes, which then react with Schiff`s reagent to produce the characteristic pink or magenta color. PAS staining can be used to detect various types of carbohydrates, such as polysaccharides, mucin, glycogen, and fungal cell wall components.
PAS staining can be applied to different types of biological samples, such as formalin-fixed, paraffin-embedded tissue sections, or frozen tissue sections. It can also be combined with other staining techniques, such as hematoxylin or methyl green, to provide contrast and highlight the nuclei of the cells. PAS staining can also be used in conjunction with diastase enzymes, which are able to break down glycogen into glucose. By treating the sample with diastase before PAS staining, the glycogen content can be selectively removed and differentiated from other carbohydrates.
PAS staining has many applications in histochemistry and histological studies, as it can reveal the distribution and localization of carbohydrates and carbohydrate-rich compounds in various tissues and organs. It can also help in the diagnosis of various diseases and conditions that affect the carbohydrate metabolism or composition of the cells. Some examples of PAS staining applications are:
- Cytology: PAS staining can help in the identification of glandular carcinomas (adenocarcinomas), which are tumors that arise from secretory epithelial cells that produce mucin.
- Pathology: PAS staining can help in the diagnosis of liver and kidney diseases, such as glycogen storage diseases, Wilson`s disease, diabetic nephropathy, and renal cell carcinoma.
- Fungal studies: PAS staining can help in the detection of fungal infections, such as candidiasis, aspergillosis, and cryptococcosis, by showing the fungal hyphae and yeast-forms in tissue samples.
- Gastrointestinal pathology: PAS staining can help in the evaluation of the mucosal lining of the gastrointestinal tract, such as the esophagus, stomach, small intestine, and colon.
- Lung studies: PAS staining can help in the assessment of lung diseases, such as pulmonary alveolar proteinosis (PAP), which is characterized by the accumulation of proteinaceous material in the alveoli.
- Skin studies: PAS staining can help in the identification of skin lesions, such as eosinophilic globoid bodies (Kamino bodies), which are associated with malignant melanoma.
- Muscle biopsies: PAS staining can help in the demonstration of glycogen content in muscle fibers, which can be useful for diagnosing metabolic disorders or muscular dystrophies.
- Enzymatic cytochemistry: PAS staining can help in the detection of granules that contain enzymes involved in carbohydrate metabolism or degradation.
In summary, PAS staining is a versatile and useful technique that can provide valuable information about the carbohydrate composition and function of biological samples. It can also aid in the diagnosis and prognosis of various diseases and conditions that affect the carbohydrate metabolism or structure of the cells.
PAS staining is based on the principle that periodic acid can oxidize some carbohydrates and carbohydrate-rich compounds to form aldehydes. These aldehydes can then react with Schiff`s reagent, a colorless compound that contains fuchsin, to produce a magenta-colored complex. The intensity of the color depends on the amount and type of carbohydrates present in the tissue sample.
The oxidation reaction of periodic acid with carbohydrates involves breaking the bonds between two adjacent carbon atoms that are not involved in the glycosidic linkage or ring closure of the monosaccharide units. This creates two free aldehyde groups at the ends of each broken monosaccharide ring. For example, periodic acid can oxidize glycogen, a polysaccharide composed of glucose units, to form multiple aldehydes .
The Schiff`s reagent is prepared by adding hydrochloric acid and potassium metabisulfite to basic fuchsin, a red dye. The sulfite ions reduce the fuchsin to form a colorless leucofuchsin, which is also known as Schiff`s reagent. When Schiff`s reagent encounters an aldehyde group, it is oxidized back to fuchsin and forms a purple-magenta complex with the aldehyde .
The PAS staining technique can detect various types of carbohydrates and carbohydrate-rich compounds, such as polysaccharides, mucins, glycoproteins, glycolipids and fungal cell wall components. These substances are usually found in connective tissues, mucus, glycocalyx, basal laminae and other structures that have important biological functions .
To enhance the contrast and visualization of the PAS-positive structures, a counterstain is often used to stain the nuclei and the background. Hematoxylin or methyl green are commonly used as counterstains for PAS staining. Hematoxylin stains the nuclei blue or purple, while methyl green stains them green. The background usually stains blue with either counterstain .
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Therefore, PAS staining is a useful technique to demonstrate the presence and distribution of carbohydrates and carbohydrate-rich compounds in tissue samples. It can also help to differentiate between different types of carbohydrates based on their staining intensity and resistance to enzymatic digestion . PAS staining can be combined with other staining methods, such as alcian blue or diastase, to further identify specific types of carbohydrates or to remove unwanted staining from glycogen .
Before performing the PAS staining, you need to prepare the following solutions and reagents:
- Periodic acid solution (0.5%): Dissolve 0.5 g of periodic acid in 100 ml of distilled water. Store in a dark bottle at room temperature. This solution is stable for several months.
- Schiff reagent: Dissolve 1 g of basic fuchsin in 100 ml of boiling distilled water. Add 1 ml of concentrated hydrochloric acid and cool. Filter the solution and add 2 g of activated charcoal. Filter again and store in a dark bottle at room temperature. This reagent is sensitive to light and air, so it should be replaced every 2-3 months or tested before use.
- Testing Schiff reagent: To test the quality of Schiff reagent, add 10 ml of 37% formalin to a small amount of the reagent. A good Schiff reagent will turn red-purple in color within a few seconds, while a poor Schiff reagent will have a delayed reaction producing deep blue-purple coloration.
- Mayer`s hematoxylin solution: Dissolve 1 g of hematoxylin in 10 ml of glycerol and 10 ml of ethanol. Add 200 ml of distilled water and 0.2 g of sodium iodate. Boil for 10 minutes and cool. Add 50 mg of citric acid and filter. Add 0.5 g of potassium alum and dissolve by heating. Cool and filter again. Store in a dark bottle at room temperature. This solution is stable for several months.
- Methyl green solution (0.5%): Dissolve 0.5 g of methyl green in 100 ml of distilled water. Filter and store in a dark bottle at room temperature. This solution is stable for several months.
You also need to have the following materials ready:
- Formalin-fixed, paraffin-embedded tissue sections, or frozen tissue sections
- Xylene or xylene substitute
- Ethanol (100%, 95%, and 70%)
- Distilled water
- Synthetic mounting medium
- Glass slides and coverslips
PAS staining is a relatively simple and quick technique that can be performed on formalin-fixed, paraffin-embedded tissue sections, or frozen tissue sections. The following steps describe the general procedure for PAS staining:
- Deparaffinize and hydrate water. If using paraffin-embedded tissue sections, remove the paraffin by immersing the slides in xylene or a xylene substitute for 10 minutes. Repeat this step twice. Then rehydrate the tissue by passing the slides through a series of graded alcohols (100%, 95%, 70%, and 50%) for 5 minutes each. Finally, rinse the slides in distilled water. If using frozen tissue sections, skip this step and proceed to step 2.
- Oxidation. Add 0.5% periodic acid solution to the slides and incubate for 5 minutes at room temperature. This step oxidizes the carbohydrates in the tissue to form aldehydes.
- Rinsing. Rinse the slides in distilled water for 5 minutes to remove excess periodic acid.
- Aldehydration. Place the slides in Schiff reagent and incubate for 15 minutes at room temperature. This step reacts with the aldehydes to form a pink or purple color.
- Washing. Wash the slides in lukewarm tap water for 5 minutes to remove excess Schiff reagent and develop the color.
- Counterstaining. Add Mayer`s hematoxylin solution to the slides and incubate for 1 minute at room temperature. This step stains the nuclei blue or green depending on the dye used.
- Wash with running tap water for 5 minutes to remove excess hematoxylin and enhance the contrast.
- Dehydrate and mount with a synthetic mount medium. Pass the slides through a series of graded alcohols (50%, 70%, 95%, and 100%) for 5 minutes each to dehydrate the tissue. Then clear the slides by immersing them in xylene or a xylene substitute for 10 minutes. Repeat this step twice. Finally, apply a drop of synthetic mount medium on each slide and cover with a coverslip.
The slides are now ready for microscopic examination and analysis.
The results of PAS staining depend on the type and amount of carbohydrates present in the tissue sample. The most common color reactions are:
- Pink or purple: This indicates the presence of glycogen, mucin, elastic fibers, basophilic granules of the pituitary gland, reticular fibers, thyroid colloids, basement membranes, bone cartilage, and other carbohydrate components. These substances react with periodic acid and form aldehydes, which then bind to the Schiff reagent and produce a pink or purple coloration. The intensity of the color may vary depending on the concentration and distribution of the carbohydrates.
- Green or blue: This indicates the presence of nuclei, which are counterstained with hematoxylin or methyl green. These dyes bind to the DNA and RNA in the nuclei and produce a green or blue coloration. The contrast between the nuclei and the cytoplasm helps to identify the cell types and structures in the tissue sample.
- Blue: This indicates the presence of background tissue elements, such as collagen fibers, muscle fibers, red blood cells, and other non-carbohydrate components. These substances do not react with periodic acid or Schiff reagent and remain unstained. However, they may absorb some of the blue light from the Schiff reagent and appear blue.
The interpretation of PAS staining requires careful examination of the tissue sample under a microscope and comparison with normal and abnormal samples. Some examples of PAS staining in different tissues are:
- Liver: In normal liver tissue, PAS staining shows a diffuse pink coloration in the cytoplasm of hepatocytes due to glycogen storage. In liver diseases, such as cirrhosis, hepatitis, or glycogen storage diseases, PAS staining may show abnormal patterns of glycogen distribution or depletion.
- Kidney: In normal kidney tissue, PAS staining shows a thin pink line along the basement membrane of the glomeruli and tubules due to glycoproteins. In kidney diseases, such as diabetic nephropathy, glomerulonephritis, or amyloidosis, PAS staining may show thickening or disruption of the basement membrane or deposition of abnormal substances.
Fungi: In fungal infections, PAS staining shows a bright pink coloration in the cell wall of fungi due to polysaccharides. The shape and size of the fungal elements can help to identify the species of fungi, such as Candida albicans (yeast-forms), Aspergillus fumigatus (hyphae), or Cryptococcus neoformans (capsule).
Applications of PAS staining
PAS staining is a versatile technique that can be used to detect various carbohydrates and carbohydrate-rich compounds in different types of tissues and cells. Some of the applications of PAS staining are:
- Cytology: PAS staining can help in the identification of tumors that secrete neutral mucins, such as adenocarcinomas. It can also be used to demonstrate glycogen in cells, such as hepatocytes, muscle cells, and erythroleukemia cells.
- Pathology: PAS staining can be useful in the diagnosis of several diseases that affect the liver, kidney, lung, skin, and other organs. For example, PAS staining can reveal glycogen storage disease, alpha-1 antitrypsin deficiency, pulmonary alveolar proteinosis, Whipple`s disease, Paget`s disease of the breast, alveolar soft part sarcoma, and ceroid lipofuscinosis .
- Fungal studies: PAS staining can highlight the fungal cell wall components, such as polysaccharides and glycoproteins, in tissue samples infected by fungi. This can help in the identification of fungal species such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans . However, PAS staining only works on living fungi, unlike other stains such as Grocott`s methenamine silver stain (GMS) that can stain both living and dead fungi.
- Gastrointestinal pathology: PAS staining can detect the presence of mucins in the gastrointestinal tract, which are secreted by goblet cells and other mucous-producing cells. Mucins are important for the protection and lubrication of the digestive system. Abnormalities in mucin production or secretion can be associated with inflammatory bowel disease, ulcerative colitis, Crohn`s disease, and colorectal cancer.
- Lung studies: PAS staining can be used to study the amorphous or granular globules of pulmonary alveolar proteinosis (PAP), which is a rare lung disorder characterized by the accumulation of surfactant-like material in the alveoli. PAS staining can also demonstrate glycogen in lung biopsy specimens of infants with pulmonary interstitial glycogenosis (PIG), which is a rare congenital disorder that causes respiratory distress and failure .
- Skin studies: PAS staining can be used to study the eosinophilic globoid bodies or Kamino bodies, which are characteristic features of Spitz nevus, a benign melanocytic tumor of the skin. PAS staining can also demonstrate basement membranes and elastic fibers in the skin.
- Muscle biopsies: PAS staining can demonstrate glycogen components in muscle tissues, which are important for energy metabolism and muscle contraction. Glycogen abnormalities can be seen in glycogen storage diseases, such as McArdle disease and Pompe disease.
- Enzymatic cytochemistry: PAS staining can be combined with diastase digestion to detect granules that contain enzymes such as alpha-amylase and alpha-glucosidase. This can help in the identification of salivary gland tumors and pancreatic tumors.
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