STAINS USED IN LIGHT MICROSCOPY – 1
Most histological sections must be stained to be studied under a light microscope.
For this purpose, numerous dye solutions and combinations of dyes have been developed. 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 were also developed and became very useful. Some dye solutions are composed of one molecule whereas others are solutions of salts in which a part of their molecule exhibits a color.
One of the most widely used staining procedure, 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 staining.
During this staining technique the sections are stained initially with a solution of hematoxylin followed by a solution of eosin (the whole staining procedure is actually more complex and will not be discussed 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 colored. 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 molecule, not even a salt, but in solution it behaves as a basic dye.
A solution of toluidine blue, methylene blue or hematoxylin stains components of cells and of the extracellular matrix in a purple/blue color.
Whats happens during the staining procedure of sections?
Most commonly, the components of the cells and extracellular matrix that contain many acidic (negative) chemical groups exhibit affinity for the (positive) basic dyes. For this reason, these cell and extracellular structures of the sections are called basophilic, meaning that they are friendly to basic dyes.
Examples of basophilic structures stained by hematoxylin and by basic dyes:
– the nuclei and the nucleoli) are rich in nucleic acids. The nucleic acids have an affinity for basic stains. For this reason the nuclei and nucleoli become stained in blue-purple by hematoxylin and by basic dyes.
– the ergastoplasm of the citoplasm (called granular endoplasmic reticulum when observed by electron microscopy) contains a lot of ribonucleic acid in its ribosomes and is a basophylic component of the cytoplasm of many cell types. It stains blue/bluish by hematoxylin.
– the extracellular matrix of hyaline cartilage contains many molecules that have acidic groups (for example sulfates) and this matrix stain blue with hematoxylin.
Acidic stains
The acidic dyes, also called acid dyes, are another important group of dyes. In the molecules of these dyes, 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-orange 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-orange by eosin.
A warning:
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 be observed under a light microscope.