Ferritin immunohistochemistry as a marker for microglia

Abstract

An immunohistochemical analysis of formalin-fixed, paraffin-embedded brain sections was performed with antisera against holoferritin and the light(L)-subunit of ferritin. Sections immunostained using anti-glial fibrillary acidic protein (GFAP), Ricinus communis agglutinin-1 (RCA-1) stain for microglia and iron stain (Berlin blue stain) were compared. The L-subunit of ferritin was purified from normal human spleen according to the modified scrapie-associated fibrils purification, and the antiserum was raised in a rabbit. Both ferritin antisera positively stained resting and, more markedly, reactive microglia, both of which were also stained with RCA-1 but not with GFAP. Ferritin-positive resting microglia were seen more abundantly in cerebral and cerebellar cortices than in white matter. The advantages of ferritin antisera over RCA-1 are as follows. (1) RCA-1 heavily stains blood vessels, while anti-ferritin does not, hence the microglial cells are more readily visualized with ferritin immunohistochemistry. (2) Reactive microglia and macrophages are more strongly stained with anti-ferritin. (3) The staining intensity of ferritin is independent of the length of tissue fixation in formalin. However, anti-ferritin is inferior to RCA-1 in staining resting microglia with a scanty cytoplasm, especially in the white matter, probably because the former recognizes cytoplasmic components, while the latter recognizes cell membrane. Iron stain only gave a reaction to microglial cells in brains with neurosyphilis and to hemosiderin-laden macrophages. Thus, in addition to RCA-1, ferritin antisera are useful as a microglia marker in formalin-fixed, paraffin-embedded sections.