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Core curriculum of Neuropathology

Cell types & networks
Staining methods
Cellular pathology

Staining methods / Staining of nerve tissue

H&E staining

This is a standard staining method used in pathology. Typically, the cytoplasm of cells is eosinophilic (acidophilic) and is stained red, whereas the nuclei and nucleoli are “hematoxylinophilic” (basophilic) and are stained blue. Other physiological and pathological structures show various staining behaviors; therefore, it is important to understand each individual structure. Nissl bodies, which are the rough endoplasmic reticulum of neurons, are basophilic, while other cytoplasmic structures are mostly eosinophilic. Axons and dendrites cannot be distinguished unless there are swelling-related changes. Astrocytes lack an eosinophilic cytoplasm and have nuclei that appear large and quite clear. When astrocytes react to tissue damage, they appear eosinophilic because their cytoplasm becomes more abundant as a result of an increase in fibrous components, which also accumulate in the nerve processes. On the other hand, oligodendroglia are smaller than astrocytes, with basophilic densely staining nuclei and a barely visible cytoplasm. The nuclei of microglia present with basophilic club-shaped terminations and are thus easily distinguished.

Nissl staining

In this method, neuronal Nissl bodies (the rough endoplastic reticulum) are stained purple using cresyl violet. The stained Nissl bodies appear aggregated and brindled. Nissl staining is convenient for measuring the density of neurons because stained cells are clearly defined and easily measured.

Luxol-fast blue (LFB) staining

LFB is a myelin sheath stain that stains phospholipids (the main constituents of the myelin sheath around nerve processes) blue. Luxor is the name of a city in Egypt where the blue dye is collected. The myelin-rich cerebral white matter is stained blue. In demyelinating diseases where the myelin sheath is broken down, the distribution of lesions can be clearly identified.

Kluver–Barrera (KB) staining

KB is the most common nervous system-specific stain used. It involves double staining with Nissl and LFB stains, and it simultaneously stains both neurons and the surrounding myelin sheath. Magnified images of stained specimens resemble those observed in LFB staining; however, because the neurons are also Nissl stained, the gray matter turns slightly blue. Nissl bodies are stained with Nissl stain and the surrounding myeli

Bodian silver staining

Bodian staining uses silver proteins, copper, and gold chloride to stain neuronal cell bodies (soma) and nerve processes dark brown. Normal and abnormal structures formed by abnormal fiber components are also stained. Argentaffin parts include areas of localized axonal swelling (spheroid), dendritic lesions, and Alzheimer’s neurofibrillary degeneration [neurofibrillary tangles (NFTs)].

Other silver stains used for nerve tissue

Other silver staining procedures such as Bielschowsky and methenamine silver staining are only performed as required. Bielschowsky staining clearly stains nerve fibers. Methenamine silver is an argyrophilic stain that enables clear visualization of amyloid components of senile plaques; immunostained images of β-amyloid proteins show similar results.

Holzer staining

The fibrous components of astrocytes are stained purple with Holzer’s crystal violet. In particular, gliosis, which is secondary scarring following nerve damage, is clearly stained. Therefore, sites that have been damaged can be macroscopically detected. Because the aniline in the stain solution is harmful, staining must be performed in a laboratory draft chamber (i.e., fume hood). Therefore, Holzer’s staining in addition to phosphotungstic acid hematoxylin (PTAH) staining can be used to detect gliosis.

Gallyas–Braak (GB) staining

In the late 1990s, GB staining gained rapid popularity in the field of neuropathy. While it is a type of argyrophilic stain, it does not stain the normal existing tissue, thereby enabling clear identification of deposits partly consisting of abnormal tau and other pathological structures. While these can be detected with H&E and Bodian staining, they were first visualized with GB staining. Nonetheless, the normal background tissue is not stained, whereas only abnormal structures are stained black; therefore, GB is convenient when examining the distribution of abnormalities. Abnormal accumulated tau proteins should be labeled.