Application Focus: New targets for immunostaining analysis of microglia

Tue, 08/01/2017 - 09:35

Microglia are resident macrophages in the central nervous system (CNS) that play roles in immune defense, inflammatory response, neurodegenerative disease and development. Identification of microglia has confounded researchers aiming to understand their biological function in the CNS, as they are molecularly and morphologically similar to other myeloid cells. For example, morphologically, microglia are indistinguishable from infiltrating blood derived macrophages.1 Additionally, microglia are commonly purified for study based on their expression of molecular markers, such as CD11bHigh and CD45Low; although, these may change in disease states.2,3

To fully understand and uncover the roles of microglia in the CNS, specific markers that accurately identify these cells are needed. Recently, investigators have embarked in efforts to elucidate specific microglial markers using advanced genomic and proteomic approaches. However, only a few of the many newly identified microglial enriched markers have been validated.

Using a combination of gene profiling and quantitative mass spectrometry analysis, Butovsky et al. investigated markers uniquely expressed by adult murine microglia.1 To accomplish this goal, the genes and proteins expressed in microglia were first compared to those from splenic monocytes. They focused on the Ly6C+ monocyte subset, known to be recruited to the CNS during inflammation. This comparison allowed investigators to identify genes and proteins enriched in microglia.

P2Y12 P2RY12 Antibody IHC-P

(Right) Immunohistochemical staining of human cerebral cortex shows strong cytoplasmic positivity in microglia.

To further refine the specificity of the identified microglial genes, investigators designed a gene-expression chip, containing microglia- and inflammatory-genes, to compare against macrophages and other immune cells. Investigators also examined the expression of microglial genes in other cells including astrocytes, oligodendrocytes and neurons. Overall, their findings allowed them to identify a series of genes, uniquely expressed in microglia and not in any other cell type tested. Thus, these genes may serve as key markers of microglial identity and provide useful targets in immunostaining or flow cytometry for the identification and purification of microglia (see Table).

Microglial unique genes Novus antibodies
Fcrls FCRL2/FcRH2 (MAB2048) (FC)
P2ry12 P2Y12/P2RY12 (NBP2-33870) (IHC)
Tmem119 TMEM119 (NBP2-30792) (IHC)
Olfml3 OLFM-L3 (NBP2-15055) (IHC, ICC/IF)
Hexb HEXB (NBP2-49211) (IHC, ICC/IF)
Tgfbr1 TGF-beta RI/ALK-5 (AF3025) (ICC)
FC, Flow cytometry; IHC, Immunohystochemistry; ICC, immunocytochemistry IF, Immunofluorescence

Continuing the quest for ideal microglial markers, more recently Bennett et al.3 developed new tools for the study of microglia. They focused on the transmembrane protein, Tmem119, which was identified as a highly-expressed protein in the CNS and limited to microglia. Investigators demonstrated that Tmem119 was developmentally regulated and stably expressed in microglia from early postnatal stages to adult stages. Using this marker to isolate microglia from murine brain, allowed them to further understand the specific gene expression profiles of nonactivated-developing microglia. Among the identified microglial specific genes, P2ry13 and Olfm13 were found to be upregulated between embryonic and adult stages. Therefore, these proteins may serve as stable markers to study microglial functions throughout development.

These studies have made significant strides in identifying novel and specific microglial markers. Specific molecular markers should facilitate the identification and isolation of microglia and further our understanding of microglial functions in various disease states.

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  1. Bennett M. L. et al. (2016). New tools for studying microglia in the mouse and human CNS. Proceedings of the National Academy of Sciences of the United States of America, 113(12), E1738–E1746.
  2. Derecki N. Cronk J. & Kipnis J. (2012). FACS of acutely isolated mouse microglia. Protocol Exchange. Nature Publishing.
  3. Butovsky, O. et al. (2014). Identification of a Unique TGF-β Dependent Molecular and Functional Signature in Microglia. Nature Neuroscience, 17(1), 131–143.

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