Figure 4

Iba-1 and CD45 markers immunostain different phenotypes of microglial cells. Immunohistochemical detection with Iba-1 antibody identified microglia both near and away from amyloid deposits. (A) Activated microglia near and around Congo red positive amyloid deposits (arrows) show increased Iba-1 expression and possess larger cell bodies with short thick processes. Typically, these activated microglia cluster together forming a cordon around amyloid deposits. (B) Microglia away from or in between amyloid deposits (arrows) differ in morphology from the previous type with respect to their smaller cell bodies and long thinner processes. These quiescent microglia typically maintain distance from each other and are actively receiving inputs about their respective microenvironments [29] (C) The microglial marker CD 45 clearly identifies only activated microglia near amyloid deposits and possesses larger cell bodies with short thick processes. CD45 antibody stained no microglia away from or in between Aβ deposits. (D) CD45 also identified many dark intensively stained round cells (arrows) which were often found isolated or in groups inside brain parenchyma or near blood vessels (E) or meninges (F). (G) Phenotypes marked by both Iba-1 and CD45 can be seen together in a double fluorescent immunostaining. Maximun intensity projection of a confocal Z-stack revealed distinct Iba-1 (green) and CD45 (red) positive cell populations pointing to simultaneously co-existing different morphological phenotypes of activated and resting microglia. Intensively stained iba-1 positive microglia (green) are seen as clumps (arrow) around a putative amyloid deposit. Some but not all microglia (red) in clumps were also CD45 positive, thus producing a yellow signal indicative of co-localization. In contrast, quiescent microglia away from putative amyloid deposits showed less intense Iba-1 (green) signal. This way it is feasible to simultaneously identify and quantify microglial cells based on their morphological (circularity) and biochemical (Iba-1 and CD45 expression) attributes. Scale bars represent 500 μm except for the confocal fluorescent image where it represents 50 μm.