Histochemical techniques to demonstrate Carbohydrates-Polysaccharides

Introduction

Carbohydrates are the most abundant and one of the four essential macromolecules, required for the survival of living beings. Structurally, these are polyhydroxy aldehydes or ketones. Carbohydrates are divided into three classes depending upon the number of forming units (aldehyde or ketone), which are monosaccharides, oligosaccharides, and polysaccharides.

These carbohydrates have extensive roles to perform inside the living organism. Monosaccharides, the simplest unit of carbohydrates, including glucose, which acts as an energy source, and amino sugars are the structural part of oligosaccharides and polysaccharides. Disaccharides like sucrose and maltose are used as sweeteners and sucrose also acts as a major source of energy in plants. Polysaccharides provide mechanical support to cells in different organisms and they also help in energy storage.

In this article, we explore polysaccharides, and histochemical techniques to demonstrate them for various research and biomedical purposes.

For the purpose of demonstration, the sample tissue should be prepared by freezing first, followed by sectioning and fixation of it.

Carbohydrate Fixation

For the best results, it is recommended to use different fixatives for different types of carbohydrates. This is elaborated in the table below:^[4] 

Histochemical demonstration of Carbohydrates

Carbohydrates which can be demonstrated by histochemical methods are polysaccharides. Polysaccharides are polymers of more than ten monosaccharides units. There are two types of polysaccharides: homopolysaccharides and heteropolysaccharides. Homopolysaccharides contain a single type of monomeric unit (example: starch, glycogen, cellulose, and chitin), while heteropolysaccharides contain two or more kinds of monomeric units (example: glycosaminoglycans and peptidoglycan).

Demonstration of Homopolysaccharide

1.     Starch

Starch is a branched polymer of D-glucose units. It is a mixture of amylose and amylopectin. They are the storage form of polysaccharides in plants. The presence of starch in tissues can be determined by an iodine test.

• Iodine Test

Principle: Reaction of iodine with the amylose in starch results in the formation of a polyiodide chain which gives deep blue color.
Materials required: Sample tissue, Iodine-potassium solution (add 0.2g iodine in 2% potassium iodide solution), distilled water, glycerin jelly, and slides.
Procedure:

• Place the section of a sample tissue in iodine-potassium for 2 minutes.
• Rinse the section with distilled water.
• Mount in glycerine jelly.^[8]

Observation: You will observe deep blue or blue-black colored starch granules in the tissue section.

2.     Glycogen

In animals, glycogen is the major storage form. It is a highly branched polymer of D-glucose units and is mostly found in the liver and the muscles.

• Carmine Method
  Principle: Carminic acid reacts with the hydroxyl group of glycogen (formation of hydrogen bonding) that results in red color glycogen.
  Materials required: Sample tissue, Hematoxylin crystal, Ferric chloride, concentrated HCl, Carmine, potassium carbonate, potassium chloride, ammonium hydroxide, absolute alcohol, methanol, distilled water, and slides.
  Reagents preparation
  Weigert’s Iron Hematoxylin:
  Solution A:0 gm hematoxylin crystals in 100 ml of 90% alcohol;
  Solution B: Add 4 ml Ferric chloride in 95 ml of distilled water followed by 1 ml of concentrated HC.

For Weigert’s Iron Hematoxylin solution, mix solutions A and B in equal parts for use.
Carmine solution (stock): Add 2.0 gm carmine, 1.0 gm potassium carbonate, 5.0 gm potassium chloride in 60 ml distilled water. Boil solution for 5 minutes; cool it down; add 20 ml of  28% ammonium hydroxide; you can store it in the refrigerator.
Carmine working solution: Mix 10 ml carmine solution, 15 ml 28% ammonium hydroxide, and methyl alcohol.
Differential solution: mix 20 ml absolute alcohol, 10 ml methanol, and 25 distilled water.
Procedure:

• Deparaffinize the section containing slide and hydrate it by using distilled water.
• Put the slide in Weigert’s iron Hematoxylin for 1 minute.
• Wash the slide under running water.
• Rinse the slide with 0.5 % HCl followed by 70% alcohol for 10 seconds.
• Wash the slide under running water for 5 minutes.
• Rinse the slide with distilled water.
• Put the slide in a working carmine solution for 30 minutes.
• Transfer the slide in differentiating solution for 3 seconds.
• Rinse the slide in 70% alcohol.
• Dehydrate the slides in graded alcohol.
• Clear the slide in xylene and mount in synthetic resin.^[5]

Observation:  You will observe glycogen granules in pink to red color.

1. Periodic acid-thiocarbohydrazide-silver proteinate reaction
   Principle: Reduction of osmium tetroxide and silver salts occurs when thiocarbohydrazide is added to the carbonyl solution.
   Materials required: Sample tissue, Paraperiodic acid (H₃IO₆), Crystalline thiocarbohydrazide (TCH), acetic acid, Triple distilled water, protargol silver proteinate, and columbia jar.
   Procedure:

• Oxidize the tissue section with H₃IO₆ and then wash it with distilled water.
• In 1% w/v TCH (dissolved in 10% v/v acetic acid), incubate the section at room temperature for 5 minutes.
• Rinse the section with 5 % and 1 % v/v acetic acid.
• Wash the section with distilled water for 3 minutes.
• Pipette out 10 ml of triple distilled water (before using, sprinkle 100 mg propargyl silver proteinate without stirring) in Columbia jar.
• Heat the staining solution at 50 °C for 5 minutes and put the section in it for 20 minutes.
• Blot the section with filter paper and then wash it with distilled water.^[2]

Observation: You will observe silver granules of glycogen in the tissue.

3.     Cellulose and Chitin

Cellulose is a linear polysaccharide of glucose monomer having B-1, 4 glycosidic linkages. This is insoluble in various organic solvents, as well as in water. This can be obtained from the by-product of various plants such as sorghum, sugarcane, wheat, and rice.

Chitin is a structurally long polymer of N-acetylglucosamine, which is an amino sugar and a derivative of glucose. It is present in the cell wall of plants and in some fungi, where it plays a role in structural support and protection under harsh conditions.

Calcofluor white staining method

Principle: Calcofluor white is a fluorochrome stain. It is non-specific in nature and stains cellulose and chitin by binding with it in the tissue environment.

Materials required: Sample tissue, calcofluor white, distilled water, fluorescence microscope, and slide.

Procedure:

• Deparaffinize the tissue section containing the slide.
• Put the slide in 1 % calcofluor white for 20 seconds.
• Wash the slide twice with distilled water.
• Mount the slide.
• Observe the slide under a fluorescence microscope.^[1]

Observation: You will observe blue-colored fluorescent cellulose under the microscope.

Note: Calcofluor white can also bind to callose, chitin and other polysaccharides.

Demonstration of Heteropolysaccharides

Heteropolysaccharides are also called heteroglycans. They are composed of two or more different units of monosaccharides. Mainly it includes glycosaminoglycans (for example- hyaluronate, chondroitin sulfate, and keratin sulfate) and peptidoglycan.

1.     Glycosaminoglycans

These are also called mucopolysaccharides or proteoglycans, which are linear molecules containing uronic acid and sulfated groups (presence of uronic acid and sulfated group at free ends make it highly acidic). It mainly includes four groups, Hyaluronate, heparin sulfate, keratin sulfate, and chondroitin sulfate. Hyaluronic acid is a non-sulfated group of glycosaminoglycans (GAG). These are components of the extracellular matrix which also give mechanical support to the cell.

These mucosubstances (acidic and non-sulfated/sulfated) can be demonstrated by various methods like Hale’s colloidal iron method, Periodic-acid-Schiff’s reaction (PAS), Alcian blue, and Metachromatic dyes.

1. Hale’s colloidal iron method
   Principle: At very low pH, carboxyl and sulfate-containing substances absorb the colloidal ferric ions. Prussian blue staining reaction then stains the absorbed ferric substance in blue.
   Materials required: Tissue section, 12% acetic acid, 2% aqueous potassium ferrocyanide, colloidal iron suspension (make a working colloidal solution by adding colloidal iron suspension in acetic acid, in equal volumes), and Perl’s solution (mix 2% ferrocyanide and 2% HCl in equal volumes).
   Procedure:

• Deparaffinize the section and rinse it with distilled water.
• Again, rinse the slides well in the 12% acetic acid.
• Put the section in a working colloidal solution for 15-20 minutes.
• Rinse the section three times with a 12 % acetic acid solution.
• Put the section in Perl’s solution for 20 minutes.
• Wash the section with distilled water to remove the extra solution.
• Counterstain the section with nuclear-fast-red for 1 minute.
• Dehydrate the section, and mount it in DPX.^[9]

Observation: You will observe the acid mucopolysaccharides stained in deep blue color, which shows the presence of glycoproteins (example GAGs).

1. Periodic-acid-Schiff Reaction
   Principle: The free hydroxyl group is oxidized by periodic acid to aldehyde. When this aldehyde group comes in contact with Schiff’s reagent, it forms a magenta-colored complex.
   Materials required: Tissue sample, Periodic acid solution (1 gm periodic acid in 100 ml distilled water), Schiff’s reagent (add 1 gm fuchsin basic in 100 ml boiling distilled water then add 2 gm sodium metabisulfite and 2 ml HCl), and hematoxylin.
   Procedure:

• Deparaffinize the section and wash it in distilled water.
• Treat the section with periodic acid for 5 minutes.
• Rinse the section well with distilled water.
• Put the section in Schiff’s reagent for 10-15 minutes.
• Wash the section well under distilled water to remove the extra chemicals.
• Counterstain the section with hematoxylin for 15 seconds.
• Wash the section again with distilled water.
• Rinse the section with alcohol.
• Clean the slide with xylene and mount it.^[7]

Observation: You will observe the section in the magenta color that shows the presence of proteoglycans.

Note: Hyaluronic acid can not be stained with PAS. Complex substances such as chondroitin and keratan sulfate also give a negative PAS test.

1. Alcian blue
   Principle: Alcian blue is a basic dye which mainly stains acidic mucosubstances that are carboxylated and sulfated by forming a salt bridge with them.
   Materials required: Tissue sample, 3% acetic acid solution, Alcian blue solution (pH 2.5) [1gm alcian blue in 100 ml of 3% acetic acid solution], Nuclear fast red solution, and xylene.
   Procedure:^[3]

• Deparaffinize the tissue and wash it under running distilled water.
• Stain the section of tissue in the alcian blue solution for 30 minutes.
• Rinse the section in distilled water to remove the extra stain.
• Counterstain the section with nuclear fast red for 5 minutes.
• Wash the section again in distilled water for 1 minute.
• Dehydrate the section, clear and mount it for observation.

Observation: Nuclei will be stained in pink to red, mucosubstances in blue color, and cytoplasm will be stained in pale pink color.

1. Iron diamine method
   Principle: Diamine stain the O-sulfate esters by oxidizing itself in the reaction with ferric chloride.
   Materials required: Sample tissue, N, N-dimethyl-m-Phenylenediamine, N, N-dimethyl-p-Phenylenediamine, HCl, Na2HPO4, absolute alcohol, xylene, and slide.

Procedure:

• Deparaffinize the slide and hydrate it in graded alcohol.
• Preheat 1 N HCl at 60 °C and hydrolyze the section in it for about 10 minutes (to remove interfering stains).
• Wash the slides in running water for 5 minutes.
• Stain the section in the diamine solution for 20-48 hours. The diamine solution should be freshly prepared.
• Dissolve 30 mg N, N-dimethyl-rn-phenylenediamine (HC1) 2 and 5 mg N, N-dimethyl-p-phenylenediamine (HC1) in 50 ml distilled water and then adjust the pH to 3.4-4.0 with 0.2 M Na2HPO4 (0.15-0.65 ml).
• Rinse the section with 95 % alcohol and then with absolute alcohol.
• Dehydrate the section in xylene and mount it in xylene.^[6]

Observation: You will observe the mucosubstances in black-brown color.

Demonstration of Glycoproteins

Glycoproteins are branched molecules containing sialic acid and fucose groups. The presence of sialic acid at the free end of the glycoproteins makes it a negatively charged compound. Most of the glycoproteins compose the integral membrane protein, where they have an essential role in cell-cell interaction.

Methods of demonstration:  All the method which are involved in the demonstration of Glycosaminoglycans (GAGs) can also be used to demonstrate Glycoproteins. Like PAS (Periodic acid Schiff) reaction, alcian blue, and Cuprolinic blue staining method.

Application of Histochemical demonstration of Polysaccharides

1. Glycogen storage disorders (GSD): In this condition, glycogen gets accumulated in the tissue because of the deficiency of an enzyme that causes the breakdown of glycogen. Anyone suffering from GSD is diagnosed by taking sample tissue from the liver or muscle cell to check the level of glycogen in it.
2. Barrett’s esophagus:  In this disease, there is an abnormal growth of the esophagus and mucin is secreted in a very high amount. This disease is studied by the histochemical demonstration of mucin by staining with alcian blue.
3. To demonstrate plant cell wall: To study the structural property of plant cells, compounds like cellulose, chitin, and pectin are stained (these compounds form the secondary cell wall of the plant).
4. Hunter syndrome: In this disease, there is an accumulation of heparan sulfate and dermatan sulfate in all body tissues. This disease can be diagnosed by demonstrating the accumulation of these compounds through histochemical methods.
5. Macular corneal dystrophy (MCD): This condition is due to the accumulation of abnormally sulfated keratan sulfate, which makes the cornea opaque. Histochemical techniques are used to examine normal and MCD cornea for the accumulation of sulfated and abnormally sulfated keratan sulfate.

Conclusion

Carbohydrates are one of the essential components of the cell that is involved in the production of the energy required for the body, the structural organization of the cell, and the mechanical strength needed by the body. Histochemical demonstration of polysaccharides has major applications in the field of research and biomedical field to study variant pathological conditions. Fixation and methods of Polysaccharides demonstration depend upon the type of carbohydrate to be demonstrated. This area is evolving as some of the new techniques that involve the use of probes and fluorescent protein having an affinity with carbohydrates have also been introduced.

References

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2. Hilda K. Lo, Theodore I. Malinin, and George I. Malinin (1987). A modified Periodic Acid-Thiocarbohydrazide-Silver Proteinate staining sequence for enhanced contrast and resolution of glycogen depositions by Transmission Electron Microscopy. The Journal of Histochemistry and Cytochemistry, 35(3), 393-398. DOI: 10.1177/35.12.3316377.
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5. Mallory, F.B. (1961). Pathological Technique: a practical manual for the pathological laboratory. New York, Hafner Publishing Co. 126-128.
6. Spicer S. S. (1964). Diamine methods for differentiating Mucosubstances Histochemically. The Journal of Histochemistry And Cytochemistry. 13(3). doi/10.1177/13.3.211.
7. Thonard J. C. & Scherp H. W. (1962). Histochemical demonstration of acid mucopolysaccharides in human gingival epithelial intercellular spaces. Archives of Oral Biology, 7(2), 125–136. doi:10.1016/0003-9969(62)90001-8.
8. ThusharaT ,Devipriya V (2017). Histochemical localization of starch, protein, lipid and lignin in the callus, field-grown and in vitro raised plants of Scopariadulcis L. International Journal of Scientific & Engineering Research, 8(9), 2229-5518.
9. Wagner J. C., Munday D. E., And Harington J. S. (1962). Histochemical demonstration of hyaluronic acid in pleural mesotheliomas. The Journal of Pathology and Bacteriology, 84(1), 73 -78. doi:10.1002/path.1700840109.