STAINS USED IN LIGHT MICROSCOPY – 1
Most histological sections must be stained before they can be studied with a light microscope. Staining increases the contrast between different tissue components, making cells and extracellular structures easier to recognize.
Many staining solutions and combinations of dyes have been developed. Some are useful because they distinguish different components of cells and the extracellular matrix (ECM), whereas others are designed to reveal particular cell types or specific cellular components. Some stains consist of a single colored molecule; others are solutions of salts in which the colored part of the molecule may be either positively or negatively charged.
One of the most widely used staining procedures is the combination of two dyes, hematoxylin and eosin. This technique is called hematoxylin and eosin staining, commonly abbreviated as HE staining. In this procedure, the tissue section is first treated with a hematoxylin solution and then with an eosin solution. The complete staining procedure is actually more complex, but the details are beyond the scope of this introduction.
Basic dyes
Let us first consider a dye in which the colored part of the molecule carries a positive charge. Such dyes are called basic dyes or cationic dyes. Examples include toluidine blue and methylene blue. Hematoxylin is chemically different: it is not itself a basic dye or a salt. However, in histological staining it behaves as a basic stain.
Basic dyes bind preferentially to tissue components that contain many acidic, negatively charged chemical groups. As a result, these components become stained blue or blue-purple with hematoxylin, for example.
Tissue components that have an affinity for basic dyes are called basophilic. The term basophilic means, literally, that they have an affinity for basic dyes.
Examples of basophilic structures include:
Nuclei and nucleoli: They contain large amounts of nucleic acids, which have negatively charged groups. They therefore bind basic stains and appear blue to blue-purple with hematoxylin and the other basic dyes already mentioned.
Ergastoplasm: This term refers to the cytoplasmic region rich in rough endoplasmic reticulum. Its ribosomes contain abundant ribosomal RNA, giving this region a basophilic character. It therefore stains blue or blue-purple with hematoxylin.
Extracellular matrix of hyaline cartilage: This matrix contains many molecules with acidic groups and therefore has an affinity for basic stains. It commonly appears blue or blue-purple after hematoxylin staining.
Acidic stains
Acidic dyes, also called anionic dyes, have a negatively charged colored portion or behave chemically as acids. Examples include eosin and orange G.
Tissue components that bind acidic dyes are called acidophilic. When the acidic dye used is eosin, they are may be described as eosinophilic.
Many components of the cytoplasm have a relatively basic character and therefore bind acidic dyes. This is also true of mitochondria and several other cellular components. Consequently, after hematoxylin and eosin staining, the cytoplasm of most cells appears pink, red, or orange, depending on the tissue and staining conditions.
Similarly, collagen fibers in connective tissue have an affinity for eosin. Because collagen is a major component of the extracellular matrix in many tissues, the matrix of many tissues appears pink to orange after HE staining.
A note of caution
The classification of dyes as acidic or basic is a useful way to understand many staining reactions, but it does not apply straightforwardly to all dyes or to all staining mixtures.
It is also important to remember that HE staining is a general-purpose stain. It provides an overall view of tissue organization but does not allow most cell organelles to be distinguished individually under a light microscope. With HE staining, structures such as the nucleus, nucleolus, and ergastoplasm can be recognized because of their staining properties. Other organelles, such as lysosomes and the Golgi complex, generally require special stains or other techniques to be visualized reliably with a light microscope.