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 specifically these structures, rather than myelin sheaths, glial cells, or synaptic vesicles.
The process requires specific sample preparation. First, prolonged fixation of the material in formalin is required, lasting at least seven days. Second, to obtain high-quality sections on a freezing microtome, the sample is pre-densified by freezing.
After preparation, the sections are treated with silver nitrate (or ammoniacal silver) solutions followed by reduction. The staining principle is that cells and fibers of the nervous system selectively acquire a black color, while the surrounding tissues stain a contrasting light brown shade. In the spinal cord section, nerve cell bodies with dark cytoplasm and a lighter nucleus are clearly distinguishable, and neuronal processes are sharply outlined.
Visualization of basophilic structures (Nissl stain)
To detect specific structures within the neuron, Nissl staining is used, 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 or Nissl substance.
As a result of staining, these structures acquire a deep blue color. When examining a spinal cord neuron, one can note the clear localization of the basophilic substance: clumps are abundant directly within the neuron cell body and are also present in some of its processes, particularly the dendrites.
Visualization of myelin using osmic acid
To study myelinated nerve fibers, the method of fixation or impregnation with osmium (osmic acid) is used. The myelin sheath has a pronounced membranous, i.e., lipid, nature, which determines the choice of reagent. The method does not target fibrillar, carbohydrate, or protein components.
The principle of action is based on the fact that the method is strictly specific for structures rich in fats (lipids). Osmic acid has the ability to dissolve in lipid droplets and membranes. Due to the property of 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 treatment, two main zones are clearly visualized: in the central part is the uncolored axial cylinder (axon), and at the periphery is the thick black myelin sheath.
Briefly: answers to topical questions
Why is the Nissl substance (tigroid) absent in axons and the axon hillock?
Nissl substance (tigroid) is absent in the axon and its base (axon hillock), which serves as an important diagnostic and morphological criterion in microscopy to distinguish the axon from dendrites. The direct physiological reason for the absence is not specified in the text, but the nature of these structures is detailed.
Tigroid substance is the protein-synthesizing apparatus of the neuron. Ultrastructurally, each clump is a stack of flattened cisternae of the rough endoplasmic reticulum with attached and free ribosomes. Basophilia during staining is provided by ribosomal RNA.
Which glial cells form the myelin sheath in the central and peripheral nervous system?
The myelin sheath is formed by different glial cells depending on the division of the nervous system.
- In the CNS, myelin sheaths are formed by oligodendrocytes.
- In the PNS, these cells are called lemmocytes / neurolemmocytes / Schwann cells.
The myelin layer consists of multiple layers of oligodendrocyte or lemmocyte membrane concentrically wrapped around the axial cylinder. For the lemmocyte, the formation of the myelin layer is described by the immersion of the axial cylinder into its cytoplasm followed by the multiple rotation of the lemmocyte around its axis.
S-O-N: Silver — Silhouette of the neuron (body and processes black), Osmium — Sheath (lipid myelin black), Nissl — Nucleoproteins/basophilia (tigroid blue).
Class and exam questions
Why does the neuronal nucleus look light during silver impregnation?
The method selectively stains the cytoplasm and processes black due to silver reduction, while the nucleus binds the metal less and remains light against the dark cytoplasm background.
Where are basophilic clumps localized in Nissl staining?
Clumps of basophilic substance (tigroid) are located directly in the nerve cell body and also extend into the proximal parts of the dendrites.
How does osmic acid stain myelin fibers?
The myelin sheath is lipid in nature. Osmic acid dissolves in lipids, and due to osmiophilia, the sheath binds osmium, turning black.
What else to study on this topic
- Morphological features of motor neurons in the anterior horn of the spinal cord.
- Histology of pseudounipolar neurons of the sensory ganglion.
- Structure of pyramidal cells of the cerebral cortex.
- Schmorl's staining method and gold salt impregnation.
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