Staining of HLA-DR, Iba1 and CD68 in human microglia reveals partially overlapping expression depending on cellular morphology and pathology

•Iba1, CD68 and HLA-DR are widely used immunohistochemical markers for human microglia.•Expression of Iba1, CD68 and HLA-DR varies with the phenotype of microglia and can therefore not be used interchangeably.•HLA-DR and CD68 overall reflect immune activation and response to tissue damage of microglia but are not always co-expressed.•Iba1 is most suited for structural studies of microglia in the absence of pathology, specifically in the grey matter. ​

HLA-DR, Iba1 and CD68 are widely used microglia markers in human tissue. However, due to differences in gene regulation, they may identify different activation stages of microglia. Here, we directly compared the expression of HLA-DR, Iba1 and CD68 in microglia with different phenotypes, ranging from ramified to amoeboid, to foamy phagocytizing macrophages, in adjacent sections immunocytochemically double stained for two of the markers. Material was used from patients diagnosed with multiple sclerosis (MS) and Alzheimer's disease (AD) patients and control subjects because together they contain all the microglia activation stages in an acute and a chronic inflammatory setting. We found a similar, yet not identical, overall expression pattern. All three markers were expressed by ramified/amoeboid microglia around chronic active MS lesions, but overlap between HLA-DR and Iba1 was limited. Foamy macrophages in the demyelinating rims of active MS lesions of MS expressed more HLA-DR and CD68 than Iba1. All markers were expressed by small microglia accumulations (nodules) in MS NAWM. Dense core AD plaques in the hippocampus were mostly associated with microglia expressing HLA-DR. Diffuse AD plaques were not specifically associated with microglia at all. These results indicate that microglia markers have different potential for neuropathological analysis, with HLA-DR and CD68 reflecting immune activation and response to tissue damage, and Iba1 providing a marker more suited for structural studies in the absence of pathology.

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