The document discusses carbohydrate staining techniques used in histochemistry. It describes four groups of carbohydrates: neutral polysaccharides like glycogen and starch (Group 1), acidic mucopolysaccharides (Group 2), glycoproteins (Group 3), and glycolipids (Group 4). The periodic acid-Schiff (PAS) reaction is the most widely used chemical technique to demonstrate carbohydrates in tissue sections. PAS oxidizes glycols to aldehydes, which are then visualized using Schiff's reagent. Other stains discussed are Alcian blue, which binds carboxyl and sulfate groups, and diastase digestion to demonstrate glycogen. Quality controls are important to ensure staining technique accuracy.

1. York College CUNY
HPMT 332
Professor Emsley
Carbohydrate Stains

2. 1.Carbohydrate
• Carbohydrates- ( hydrated carbon) are important organic compounds that include
sugars, starch, cellulose and polymers that are mostly linked to proteins.
• Carbohydrates are defined chemically as ketone or aldehyde derivatives of
polyhydroxy alcohols and they may be classified as monosaccharides ( mono 1
saccharide: sugar unit.) oligo: few, 2:10) or polysaccharides: ( poly: many)
• Glucose- is the only monosaccharide found in the body in any demonstrable
quantity. Glucose is extremely soluble in aqueous solution and has a small molecular
size it cannot be demonstrated in tissue sections.
• Mucosubstances - Collective term that includes polysaccharides, glycoproteins and
proteoglycans.
• Glycogen – a polymer of glucose is the form in which carbohydrates are stored in
humans in the liver and skeletal muscles. When the cells need energy the stored
glycogen is readily broken down by the body into glucose.
• Because this enzymatic breakdown may also occur after death, if autopsy tissues are
the source for glycogen demonstration, prompt fixation has to be done.
• Glycogen is relatively insoluble in aqueous solution so it can be demonstrated in
tissue sections.

3. Group 1-Neutral
Polysaccharides( nonionic
homoglycans)
• 1. Glucose – containing: glycogen, starch,
cellulose.
• 2. N – acetyl- glucosamine – containing
chitin.
• This group gives a vey positive PAS
reaction and a negative reaction with other
carbohydrate stains ( alcian blue, colloidal
iron, mucicarmine)

4. Group II: Acid
Mucopolysaccharides ( anionic
heteroglycans)
• 1. Carboxylated ( COOH) : hyaluronic acid, found in
connective tissues and umbilical cord.
• 2. Sulfated ( OSOH) and carboxylated ( COOH)
• Chondroitin sulfate A ( chondroitin-4-sulfate)
• Chondroitin sulfate C ( chondroitin-6-sulfate), found in
cartilage, chondrosarcomas, cornea, and blood vessels
• Chondroitin sulfate B ( dermatan sulfate) found mainly in
skin, also in connective , aorta, and lung
• Sulfated only (COOH - free): human aorta and bovine
cornea.
• All polysaccharides in this group are acidic ( anionic) are
PAS negative, but stain with alcian blue, colloidal iron
and mucicarmine.

5. Group III- Glycoproteins (mucins,
mucoproteins, mucosubstances
• 1. Neutral : ovimucoid ( egg white) mucin in stomach,
Paneth cell granules
• 2. Carboxylated ( COOH) sialoglycoproteins that contain
sialic acid but no sulfate
• Sialomucins found in submaxillary gland mucin, small
intestine mucins, fetal mucins, the upper part of colonic
crypts, and human sublingual gland
• Serum glycoproteins
• Blood group substances
• 3. Sulfated and carboxylated: sialoglycoproteins- both
sialic acid and sulfate in sheep and humans mucin
• Potentially but not necessarily PAS positive

6. GroupIV: Glycolipids
• 1 Cerebrosides: fatty residue bound to a
carbohydrate structure
• 2. Phosphatides: PAS positive,
noncarbohydrate – containing lipids
including lecithin, cephalin and
sphingomyelin.

7. Histochemistry- Special Staining
Techniques
• PAS ( Periodic Acid Schiff ) reagent is prepared by
treating basic fuschin ( pararosaniline) with sulfurous
acid. Reduction cause the loss of the quinoid structure
and masking of the chromophores. A colorless
compound often referred to as leucofuschin, is formed.
Following the Schiff reaction, wash in running water
causes the loss of bound sulfurous acid group.
Mrtabisulfite rinses remove excess Schiff reagent and
prevent false coloring of tissue elements because of
oxidation of any absorbed reagent.
• Quality Control
• A section of kidney is most sensitive control.
• See page 137-139

8. Test for Quality of Schiff Reagent
• Place 10 ml of 37% to 40 % formaldehyde
in a beaker or flask.
• Add a few drops of Schiff reagent.
• If the solution rapidly turns reddish purple,
it is good.
• If the reaction delayed and the resultant
color is a deep blue-purple, the solution is
breaking down.

9. Starch
• Plant polysaccharide which has the same
general chemical structure as glycogen.
• The difference is in size, branching and
conformation of its molecules.
– Unbranched form of starch is amylose
– Branched form of starch, which is similar to
glycogen, is amylopectin.

10. PAS reaction with Diastase
Digestion
• Purpose- Demonstration of glycogen in tissue section
• Principle
• This is a very sensitive histochemical method for
glycogen. Diastase and a- amylase act on glycogen to
depolymerize it into smaller sugar units ( maltose and
glucose) that are washed out of the section.
• Quality control 2 sections of liver must be used.
• 1 labeled with and the other without. Cervix( including
both endocervix and ectocervix is also a good control.

11. Alcian blue
• This dye binds to carboxyl and sulfate
ester groups at pH2.5.
• An advantage of Alcian blue over most
other cationic dyes is that it is not
extracted from stained sections by water,
alcohol, weak acids or solutions of other
dyes used for counterstaining.

12. Chemical Methods
• The chemical technique most extensively
used in carbohydrate histochemistry is the
periodic acid-schiff (PAS) reaction.
• PAS-method – sections treated with
periodic acid which oxidizes glycols to
aldehydes.
• The aldehydes become visible by reaction
with Schiff’s reagent.
• Periodic acid HIO4.2H2O used as 1%
aqueous solution.