1. Identification of Intercellular Structures and Collagen Fibers
For a detailed study of the fibrous framework of internal organs and the reliable differentiation of collagen from other tissue elements (such as muscle tissue or epithelium), standard simple stains are ineffective. Complex multicomponent mixtures are used for these purposes in histology.
Mallory Stain This method is based on the chemical mixture of three active components simultaneously: acid fuchsin, aniline blue, and orange G. As a result of this complex tissue processing, structures acquire contrasting and easily distinguishable shades:
- Collagen fibers selectively stain a rich dark blue.
- Other structures (particularly muscle elements) acquire various dark red or orange tones.
In histological practice, the Mallory stain is actively used to identify layers of loose connective tissue inside various organs. A characteristic example is a skeletal muscle cross-section: the muscle fibers themselves appear red, while the connective tissue septa between them appear dark blue. Additionally, this method is successfully used for the complex differentiation of endocrine cells in the adenohypophysis.
van Gieson Stain This second classic differential technique uses a mixture of just two components: picric acid and acid fuchsin. When stained with the van Gieson method:
- Collagen fibers turn bright red.
- Other elements (muscle tissue and epithelial cells) stain a contrasting yellow.
2. Silver Impregnation of Reticular Fibers
Specific reticular fibers form a thin and robust supportive network (stroma) in many organs. Standard dye mixtures are not suitable for their visualization, which is why silver impregnation is used.
The principle of this chemical technique is based on argyrophilia—the pronounced affinity of certain microscopic structures for silver ions. The staining mechanism relies on the biochemical composition of the fibers themselves: they are extremely rich in specific sulfate groups to which silver binds tightly.
The process proceeds in several consecutive stages:
- The histological specimen is treated with a specialized ammoniacal silver solution.
- Reducing agents are applied to precipitate pure metallic silver directly onto the fibers.
As a result of this histochemical reaction, reticular fibers stain a rich black, forming a clearly visible network. A classic example of this technique is examining a lymph node, where the dense stroma of black reticular fibers is clearly visualized.
3. Staining of Elastic Elements
For the microscopic examination of elastic structures, which provide tissue resilience, two main approaches are used: selective and differential.
A. Orcein Stain (Selective Method) Orcein specifically identifies elastic elements. When used, elastic fibers and membranes acquire a rich cherry-red color. All other tissue elements form a pale pink background. A classic specimen for this technique is the tunica media of the aorta, where thick, wavy, concentrically arranged fenestrated elastic membranes are revealed.
B. Picrofuchsin and Hematoxylin Stain (Differential Method) This approach allows several types of fibers and cellular structures to be clearly distinguished on a single histological slide. With this combined staining:
- Elastic fibers stain yellow.
- Collagen fibers appear red.
- Cell nuclei become purple.
An excellent example is the ligamentum flavum (yellow ligament), in cross-section under a microscope clearly showing robust bundles of yellow elastic fibers and interspersed fibrocytes with purple nuclei.