Histology students must have some understanding of how histological specimens are prepared. This rudimentary understanding of tissue fixation and staining will enable students to understand the appearance of the finished product they will be studying.
Histological Tissue Preparation
The preparation of tissue for histological examination involves several steps:
- Fixation: Tissue specimens are preserved in a variety of fixatives. The type of fixative chosen is determined by the type of tissue or organ to be studied and the various components found within that tissue or organ.
- Embedding: After the tissue has been fixed it is firmly embedded into a medium that allows sectioning of the specimen at the desired thickness. Routine histological preparations are sectioned at a thickness of between 4 and 10 μm.
- Staining: An unstained specimen typically does not possess sufficient contrast to enable someone to adequately distinguish organ, tissue, and cellular characteristics. Therefore tissues will be stained with some form of organic and inorganic compounds (histological stains) that will enable adequate differentiation of tissue components and cellular detail.
Histological Stains
Although most of the photomicrographs contained within this text are of specimens stained with hematoxylin and eosin (H & E), students of histology may encounter a wide variety of stains. The most commonly encountered stains and their characteristics are listed below.
Azan
Although typically considered a connective tissue stain, this stain may also be used to demonstrate the fine cytological detail of epithelial tissue. Nuclei of epithelial tissue will stain bright red, whereas collagen of the basal lamina will stain blue. If Azan is used with other tissues, mucin will stain blue; muscle and red blood cells will stain orange.
Hematoxylin & Eosin (H & E)
This is the most commonly encountered histological stain. Hematoxylin, a base, stains acidic cellular structures, including nuclei, ribosomes, and rough endoplasmic reticulum (RER) a blue to purplish-blue color. Eosin, an acid, stains predominantly basic structures pink or red. Such structures include membranous structures within a cell. Typically a cell that is actively synthesizing proteins will stain deeply basophilic, whereas a “resting” protein-synthesizing cell will stain eosinophilic. In addition, a cell with a large number of membranous components (such as the proximal convoluted tubule cells of the kidney) will stain eosinophilic.
Masson’s Trichrome
This stain is typically used with connective tissue and produces three staining reactions: cytoplasm, muscle, erythrocytes, and keratin are stained red; basophilic structures (i.e., nuclei) are stained blue; and collagen is stained green.
Nissl and Methylene Blue Stains
A basic dye commonly used to demonstrate RER within neuronal somas.
Periodic Acid-Schiff Reaction (PAS)
Periodic Acid-Schiff Reaction (PAS) staining is a form of histochemical staining. Histochemical staining is used specifically to stain certain components of cells or tissues. PAS specifically stains complex carbohydrates a deep red color. PAS-positive components include cartilage, mucin of goblet cells, the basal lamina of renal tubules, and the brush borders of renal tubular cells.
Reticulin Stain
This stain most clearly demonstrates reticular fibers. When this stain is used, reticular fibers will stain black or dark blue.
Silver or Gold Impregnation
These “heavy metal” stains are typically used with nervous tissue to demonstrate cellular processes (axons and dendrites). Cells and cellular processes will appear back, brown, or golden when stained with either of these heavy metals.
Sudan Black and Osmium Stains
These brownish-black stains are is used to demonstrate lipidcontaining structures, such as the myelin covering of neurons.
Wright Stain
This stain is commonly used for bone marrow and blood smears. With this stain, nuclei will be purple and cytoplasm will be a light blue. Depending on their composition, cytoplasmic granules will stain variably (usually purple, pink, or unstained).
