Histochemistry. Demonstration of carbohydrates, lipids, proteins, nucleic acids

Histochemistry demonstrates a class of molecule in a section, rather than simply making the tissue visible. Each of the four classes has its own method.

Carbohydrates: the PAS reaction, purple or magenta. Lipids: dyes that are themselves soluble in lipid, the Sudan dyes, plus osmium tetroxide for unsaturated lipids. Proteins: methods directed at amino acids, and immunocytochemistry. Nucleic acids: the Feulgen reaction for DNA, the Unna-Brachet method to tell DNA and RNA apart, and hematoxylin and eosin in routine use.

Behind all of it sits one rule: nucleic acids are negatively charged, therefore basophilic, therefore they take up basic dyes.

Polysaccharides can be demonstrated by the PAS reaction, which stands for Periodic Acid-Schiff. It stains carbohydrate-rich tissues.

The mechanism is in two parts. The periodic acid breaks up the bonds between adjacent carbons. That allows the Schiff reagent to react, and the reaction gives a distinctive purple or magenta colour.

The PAS reaction is used to demonstrate:

• glycogen;
• mucus;
• glycoproteins;
• glycosaminoglycans.

A worked example: a positive PAS reaction appears at the lumen of an intestinal cell, because the microvilli there contain glycoproteins. Cell surface glycoprotein and mucin both have a high content of oligosaccharide and polysaccharide, which is what the reaction is detecting.

Lipids are best revealed with dyes that are themselves soluble in lipids. The dye does not bind chemically; it simply dissolves into the fat and stays there.

Three Sudan dyes are used, each giving its own colour:

Unsaturated lipids are demonstrated with osmium tetroxide.

Proteins are demonstrated by methods directed at their amino acids.

The more specific method is immunocytochemistry, a technique used to determine the localisation of specific proteins or antigens. Rather than detecting protein in general, it detects one named protein, using an antibody raised against it.

Nucleic acids carry a negative charge, which makes them anionic and therefore basophilic. Everything below follows from that one property: a basophilic molecule has an affinity for basic dyes.

DNA can be identified using the Feulgen reaction.

It is based on the reaction of Schiff's reagent with the aldehyde groups in the deoxyribose molecule, and it produces a magenta colour.

Because the reaction is directed at deoxyribose specifically, it stains DNA and not RNA.

The Unna-Brachet method is a technique using two basic dyes for the demonstration and differentiation of DNA and RNA.

• Methyl green stains DNA green.
• Pyronin binds to the negatively charged RNA, staining it red or pink.

One preparation therefore separates the two nucleic acids by colour, which no single dye can do.

Hematoxylin and eosin is the commonly used combination, with eosin acting as the counterstain.

Hematoxylin stains DNA in the cell nucleus, so the DNA-rich and RNA-rich portions of the cell come out dark blue or purple. Eosin stains the other cytoplasmic structures, and collagen, pink.

This is the general rule the individual methods are instances of.

Basophilic compounds are anionic, negatively charged, and react with basic dyes such as toluidine blue and methylene blue. The basophilic components of the cell are DNA, RNA and glycosaminoglycans.

Acidophilic compounds are cationic, positively charged, and react with acidic dyes such as eosin and acid fuchsin. The acidophilic components of the cell are mitochondria, secretory granules and collagen.

Elastic fibres are demonstrated with Weigert's resorcin fuchsin stain, which uses orcein to stain the elastic fibres.