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Core curriculum of Neuropathology

Cell types & networks
Staining methods
Cellular pathology

Staining methods / Immunohistochemical staining


Ubiquitin is a small protein consisting of 76 amino acids. It is found in all cells of higher life forms and plays a major role in the maintenance of the body by breaking down various proteins through the ubiquitin–proteasome system. Ubiquitin binds to target proteins (ubiquitination), which are then immediately broken down by proteasome. Target proteins to be broken down include normal proteins found in the body and abnormally aggregated denatured proteins. In general, in patients with neurodegenerative diseases, abnormally aggregated proteins are stained by antiubiquitin antibodies. These include several pathological structures, including Alzheimer’s NFTs, Lewy bodies, and skein-like hyaline, glial cytoplasmic, and ubiquitinated inclusions in the dentate gyrus in amyotrophic lateral sclerosis (ALS) with dementia. In addition, several small punctate structures called ubiqutinated dot-like structures (ubiquitin deposits) are stained with antiubiquitin antibodies in the white matter even in the healthy brain and are used as an internal control for immunostaining.

Phosphorylated tau

Tau is a microtubule-associate protein involved in the stabilization and bonding of microtubules and other cytoskeletal proteins. Tauopathy is a disease in which the phosphorylation and insolubilization of tau causes deposition within the cell and neurodegeneration. Screening for the class of tauopathy can be performed by immunostaining with phosphorylation-resistant tau antibodies, which facilitates differential diagnosis. Depending on the site of phosphorylation, several monoclonal antibodies are available for phosphorylation-resistant tau antibodies; these include Alz50, AT8, and PHF1. Phosphorylation-resistant tau antibodies test positive for pathological structures containing phosphorylated tau deposits, including Alzheimer’s NFTs, Pick’s bodies, tuft-shaped astrocytes, astrocytic plaques, and glial coiled bodies. Furthermore, there are six tau isoforms with either three or four microtubule-binding sites. The former are called 3-repeat tau, while the latter are called 4-repeat tau. It has recently been discovered that in tauopathy, there are significant deposits of both 3- and 4-repeat tau.


α-Synuclein is a soluble protein consisting of 140 amino acids in the presynaptic terminal that becomes insoluble by phosphorylation. As such, pathological structures that are immunohistologically positive for phosphorylation-resistant α-synuclein include Lewy bodies and Lewy-related neurites, which are pathological markers for Lewy body disease, and glial cytoplasmic inclusions (GCIs), which are important for the diagnosis of multiple system atrophy. The term synucleinopathy has been proposed to encapsulate these diseases. In addition, because α-synuclein is located within the presynaptic terminal, when antibodies for synapse-related proteins, including synaptophysin and SNAP25, are used, the cerebral cortex and other gray matter are evenly stained.


Antipolyglutamine antibodies are obtained from the TATA box-binding protein (TBP) and stain inclusions pathologically characteristic of triplet repeat or polyglutamine diseases, in which the hyperextension of CAG triplet repeats causes the expanded polyglutamine tract to form protein aggregates. Inclusions within the neuronal cytoplasm and nuclei in polyglutamine diseases such as Huntington’s chorea, Machado–Joseph disease, and dentatorubropallidoluysial atrophy are also labeled. Because ubiquitinization occurs in the cytoplasm and nuclei, these structures are comparably stained with antiubiquitin antibodies.


Senile plaques can be clearly detected with anti-β-amyloid antibodies. However, senile plaques are easily distinguished with H&E and other silver stains; therefore, staining with β-amyloid antibodies is rarely required.


The cytoskeleton includes neurofilaments, microtubules, and microtubule-associated proteins (MAPs). Neurofilaments consist of three subunits with different molecular masses, and antibodies exist for the detection of phosphorylated neurofilaments (2F11). Abnormal aggregation of phosphorylated neurofilaments is observed in cells located in the anterior horn of the spinal cord in ALS. MAPs also have several subunits; among these, MAP-2 is neuronal specific and is found in neuronal cell bodies (soma) and dendrites.

Substances that show chemical properties

Immunostaining using antibodies for calbindin-D, parvalbumin, and calretinin, which are calcium-binding proteins, clearly stains small inhibitory neurons in the cerebrum and cerebellar Purkinje cells. Anti-γ-aminobutyric acid (GABA) and antiglutamate decarboxylase (GAD) antibodies label GABAergic inhibitory neurons, while antibodies for serotonin and tryptophan hydroxylase label serotonergic neurons and those for tyrosine hydroxylase stain catecholamine-containing neurons.

The cytoskeleton of glial cells

The fibers of intermediate filaments located in glial cells and astrocytic soma and dendrites, in particular, are made up of glial fibrillary acidic proteins (GFAPs). Anti-GFAP antibodies clearly label pathological changes in astrocytes, including hypertrophic, fibrous, and dysplastic astrocytes. In addition, while antibodies for vimentin, an intermediate filament present in immature astrocytes, exhibit similar stainability, this protein is more abundant in dysplastic astrocytes resulting in strong staining. Antibodies specific to the cytoskeleton of oligodendroglia have not yet been popularized. Furthermore, ependymal cells test positive for anti-GFAP and antivimentin antibodies.

Substances that show chemical properties

Astrocytes are stained with anti-S-100, antiglutamate synthetase, and antiglial glutamate transporter antibodies. Oligodendrocytes are stained with antigalactocerebroside (GAL-C), anticarbonic anhydrase II (CAII), and anti-transferrin antibodies.

Antibodies that label the myelin sheath

Antibodies against myelin-associated glycoprotein and myelin basic protein label the myelin sheath; however, on a diagnostic level, this staining is of little value.