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Core curriculum of Neuropathology

Cell types & networks
Staining methods
Cellular pathology

Staining methods / Other staining methods

Periodic acid–Schiff (PAS) staining

PAS staining is generally used to detect glucides, glycogen, mucus, and fungus, which are stained red. PAS is useful when detecting substances accumulated as a result of glycogenosis and other metabolic abnormalities. In addition, phospholipids and glycolipids are stained red to purplish–red.

Toluidine blue staining

Toluidine blue staining is used for thick slices of Epon blocks used for electron microscopy. However, in the qualitative diagnosis of the cerebral nervous system, this method detects acid mucopolysaccharides using metachromatic properties. In other words, while toluidine blue is originally a blue pigment, the ability to stain acid mucopolysaccharides red enables the detection of accumulated acid mucopolysaccharides within the brain. This staining method can be used to detect accumulated substances in metachromatic leukodystrophy.

Congo red staining

Amyloid deposits, including amyloid deposits in senile plaques and vascular walls in amyloid angiopathy, are stained red with Congo red. As seen with methenamine silver, amyloid components are stained blackish–brown, and the staining behavior is similar to that in case of immunostaining using antibodies for β-amyloid proteins.

Fat staining

The staining of lipids is generally called “fat staining.” Lipids include glycerides, phospholipids, glycolipids, and fatty acids. While various lipid stains show slightly different color tones, they all exhibit similar stainability. Sudan III stains red to yellowish–red, Oil Red O stains red to reddish–orange, and Sudan Black B stains black. Glyceride is stained red, while other lipids are stained blue. For these lipid stains, frozen formalin-fixed sections are used instead of paraffin-embedded sections. The purpose of staining lipids of the nerve tissue is to examine the properties of abnormal deposits, e.g., those found in metabolic abnormalities, and to indirectly detect macrophages (lipid phagocytes) that engulf myelin sheath remnants. The latter targets macrophage infiltration to search for the presence of very mild lesion denaturation. In addition, PAS stains phospholipids and glycolipids red to purplish–red, while LFB stains phospholipids blue.

Azan staining

The fibrous connective tissue collagen, hyalinization of vascular walls, and fibrin are stained blue using this method. Azan staining is mainly used to detect structural changes in mesenchymal cells in the brain. Masson’s trichrome staining also stains collagen blue.

Elastica van Gieson staining

As its name suggests, elastica staining stains elastic fibers black or blackish–brown. Because the elastic laminae of the vascular walls are stained, this method is often used to assess vascular lesions. In addition, van Gieson collagen fiber staining is often performed simultaneously. In this method, collagen fibers are stained red, whereas muscle fibers and cytoplasm are stained blue; thus, they can be detected simultaneously.

Berlin blue staining

In this method, iron (III) is stained blue; thus, hemosiderosis in old hemorrhagic lesions can be detected. Accordingly, iron (II) is stained blue with Turnbull’s blue.

Von Kossa staining

Using this method, calcium and sodium are stained black, and the detection of calcification and pseudocalcification of the vascular walls in the brain tissue is enabled.

PTAH staining

In general, fibrous components in thrombi formed within blood vessels are stained blue to bluish–purple with PTAH stains. Consequently, this method is used to confirm thrombi when disseminated intravascular coagulation is suspected. Alternatively, glial fibers are also stained blue to bluish–purple; therefore, PTAH may be used instead of Holzer’s stain to detect gliosis. PTAH staining is more commonly used in the US and Europe. In addition, muscle fibers are stained blue.

Timm staining

Timm staining is a unique method that stains zinc black to blackish–brown. It is used to assess hippocampal sclerotic lesions in temporal lobe epilepsy. In hippocampal sclerosis, mossy fibers (i.e., granule cell axons of the dentate gyrus) and many axonal terminals appear to sprout into the molecular layer of the dentate gyrus, suggesting a relationship with intractable epilepsy. Because mossy fibers contain zinc, this method enables the visualization of the sprouting phenomenon as a thick black to blackish–brown strip in the molecular layer of the dentate gyrus. Specimens are fixed in a hydrogen sulfide-saturated alcohol solution instead of formalin; therefore, proper care is required at the time of sampling.