STAINS USED FOR LIGHT MICROSCOPY – 1
A little bit on some types of dyes
Most histological sections must be stained in order to allow them to be studied under a light microscope.
For this purpose, numerous dye solutions and dye mixtures have been developed over time. The ones that became most commonly used are those that distinguish well the various components of cells as well as of the extracellular matrix (ECM). Many dyes that demonstrate specific cell types or specific cell components became also very useful. Some dye solutions are composed of single molecules whereas others are solutions of salts in which part of their molecule exhibits a color.
One of the most widely used staining techniques, which will be briefly discussed ahead, combines two dyes called hematoxylin and eosin. This technique is called Hematoxylin and eosin staining , very commonly abridged as HE.
When the HE staining is performed, the sections are usually stained initially with a solution of hematoxylin and thereafter with a solution of eosin (but the whole staining procedure is more complex and will not be explained here).
Basic stains
Lets us imagine a solution of a dye made of salts in which their cation (i.e. the positive part of the salt) is endowed with color. Chemically speaking, these dyes have a basic nature. Examples of cationic or basic dyes: toluidine blue and methylene blue. Hematoxylin, is not such a kind of substance, not even a salt, but in solution it behaves as a basic dye.
A solution of toluidine blue, of methylene blue or of hematoxylin stains components of cells and of the extracellular matrix in a purple/blue color.
Whats happens while staining sections: most commonly, the components of the cells and extracellular matrix that contain many acidic (negative) groups exhibit affinity for the (positive) colored cations of basic dyes. For this reason, these cell or extracellular components are called basophilic, meaning that they are friendly to basic dyes.
Examples of basophilic structures:
– the cell nuclei (as well as their nucleoli) are rich in nucleic acids, which have many acidic groups. The nucleic acids have an affinity for basic stains. For this reason the nuclei and nucleoli of cells in histological sections become stained in blue-purple by basic dyes (such as toluidine blue and methylene blue) and also by hematoxylin and are therefore called basophylic structures.
– the ergastoplasm (the granular endoplasmic reticulum observed by electron microscopy) contains a lot of ribonucleic acid and is basophylic component of the cytoplasm.
– the extracellular matrix of cartilage contains many molecules that have acidic groups (mainly sulfates).
Acidic stains
The acidic dyes, also called acid dyes, are another important group of dyes. In these stains, the (negative) anionic portion of the salt is colored or it is a molecule that behaves as an acid. Examples: eosin, orange G.
Components of sections that are stained by acidic dyes are called acidophilic or eosinophilic structures.
Examples of acidophilic structures:
– the cytoplasm is aproximately basic as well as the mitochondria.
For this reason, the cytoplasm of most cells stains pink-red by eosin after HE staining.
Also the collagen fibers of the connective tissue. For this reason the extracellular matrix, which in most tissues contains a lot of collagen protein, is stained pink by eosin.
Some warnings:
The classification of acidic and basic dyes mentioned above does not apply to all dyes and dye mixtures.
In addition, HE staining is generic and does not distinguish the components of cells known as organelles, except for the nucleus, nucleolus and the ergastoplasm. Other specific organelles (lysosomes, Golgi complex, etc.) need to be treated with special stains or techniques in order to become observed under a light microscope.