Silver Nitrate Impregnation
This method is used to visualize nerve cell bodies (neurons) and their processes. It is important to note that the technique targets these specific structures rather than myelin sheaths, glial cells, or synaptic vesicles.
The process requires specific tissue preparation. First, prolonged fixation in formalin is necessary, lasting at least seven days. Second, to obtain high-quality sections on a freezing microtome, the sample is pre-embedded or frozen for firmness.
Following preparation, the sections are treated with silver nitrate (or ammoniacal silver) solutions followed by reduction. The principle of staining is that the cells and fibers of the nervous system selectively turn black, while the surrounding tissues take on a contrasting light brown hue. In a spinal cord cross-section, neuronal cell bodies with dark cytoplasm and a lighter nucleus are clearly distinguishable, and neuronal processes are distinctly outlined.
Visualization of Basophilic Structures (Nissl Staining)
To detect specific structures within the neuron, the Nissl staining method is applied, which differs histologically from the Golgi, Weigert, or routine hematoxylin and eosin stains. Toluidine blue is used as the reagent.
The principle of the method is based on the dye actively binding to moderately basophilic compounds. The main goal is to visualize clumps of basophilic substance, also known as tigroid substance or Nissl bodies.
As a result of staining, these structures acquire an intense blue color. When examining a spinal cord neuron, the distinct localization of the basophilic substance can be observed: clumps are present in large numbers directly within the neuronal cell body and are also found in some of its processes, specifically in dendrites.
Myelin Visualization Using Osmium Tetroxide
To study myelinated nerve fibers, osmium fixation or impregnation (osmic acid) is used. The myelin sheath has a prominently membranous, i.e., lipid nature, which determines the choice of reagent. The method does not target fibrillar, carbohydrate, or protein components.
The mechanism is based on strict specificity for structures rich in fats (lipids). Osmium tetroxide has the property of dissolving in lipid droplets and membranes. Due to osmiophilia (the ability to bind osmium), the lipid-containing myelin sheath acquires a dark, black color.
In a cross-section of a nerve fiber after such processing, two main zones are clearly visualized: in the central part lies the unstained axon, and on the periphery—the thick, black myelin sheath.