What are microglia?

Microglia are specialized macrophages in the brain and spinal cord. They are a type of glial cell and are the primary resident immune cells of the CNS (central nervous system). Resident microglia are central to the brain’s innate immune system, performing immune surveillance, phagocytosis, and maintaining homeostasis. Microglia function includes responding to injury, clearing debris, and supporting neural development and repair. They exhibit different activation phenotypes, primarily classified as M1 (pro-inflammatory) and M2 (anti-inflammatory), which are relevant to inflammation and tissue repair.

Microglia also play a crucial role in brain development, including synaptic pruning and neural circuit formation, supporting healthy brain maturation. In addition to maintaining homeostasis and facilitating repair, microglia are involved in neurodegenerative diseases, such as Alzheimer's and Parkinson's, and contribute to their progression.

Due to the shared lineage of microglia and macrophages, many markers are common to both cell types. However, microglia originate from embryonic hematopoietic stem cells and are distinct from peripheral immune system cells and other myeloid cell populations that can infiltrate the CNS. The distinction between resident microglia and infiltrating myeloid cells is often made using specific markers such as TMEM119 and P2RY12, which help identify the microglial cells within the CNS. Moreover, microglia are the primary resident brain immune cells, distinct from other glial and peripheral immune system cells.

TMEM119

TMEM119 is a cell-surface protein, which is a specific microglial marker in both mouse and human samples. TMEM119 is a transmembrane protein considered a highly specific marker of microglia, distinguishing them from other immune cells. TMEM119 is highly expressed in human CNS resident microglia. As part of the cluster of differentiation (CD) system, TMEM119 is used alongside other microglia markers to identify and characterize these cells accurately. This marker is not expressed by macrophages or any other immune or neural cell types, so it has a considerable advantage over other microglial markers. Transcription factors, such as PU.1, are also important for defining microglial identity in addition to surface markers.

Figure 1. Immunohistochemistry (Frozen sections) - Anti-TMEM119 antibody [28-3] - Microglial marker (ab209064).

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CD11b and CD45

A combination of CD11b and CD45 labeling can be used to differentiate microglia from macrophages. These markers are also commonly used to distinguish resident microglia from other myeloid cells, such as infiltrating immune cells, peripheral macrophages, and monocyte-derived macrophages, particularly during neuroinflammation.

In the central nervous system (CNS), resting microglia are commonly identified as CD11b^hi and CD45^low, while macrophages are identified as CD11b^hi and CD45^hi. Perivascular macrophages and infiltrating cells can be distinguished from microglia using these markers, as well as additional markers such as CD44 and Siglec-H, which are uniquely expressed on infiltrating cells. Colony-stimulating factor 1 (CSF1) and macrophage colony-stimulating factor (M-CSF) play essential roles in regulating the proliferation and activation of microglia and macrophages, which can affect the expression of these markers.

Figure 2. (A) Multiplex immunohistochemistry - Anti-CD11b antibody [EPR1344] (ab133357). (B) Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] (ab40763)

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Iba1 (ionized calcium binding adaptor molecule 1)

Ionized calcium-binding adaptor molecule 1 (Iba1) is a microglial and macrophage-specific calcium-binding protein involved with membrane ruffling and phagocytosis in activated microglia. Iba1 is upregulated during microglia activation and is key in the clearance of apoptotic cells by microglial cells. Iba1 is widely used as a marker to study microglia activation and to observe the morphological changes that microglial cells undergo during activation. Iba1 is particularly useful for identifying reactive microglia and assessing microglial activation, as its expression increases in response to pathological stimuli.

Activation of microglia is associated with increased production of reactive oxygen species (ROS), which contribute to inflammatory responses. It is important to note that rodent microglial protein expression and rodent microglia-expressed molecules can differ from those in human microglia; however, Iba1 is commonly used in mouse, rodent, and human studies to facilitate cross-species comparisons.

Figure 3. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] (ab178846).

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CX3CR1

CX3CR1 is the fractalkine receptor found on the surface of both microglia and macrophages in the CNS, where it responds to CX3CL1 (fractalkine) secreted by neurons. CX3CR1 is a cell surface glycoprotein and transmembrane protein that mediates microglia migration and adhesion during neuroinflammatory processes. In addition, CX3CR1 is involved in intracellular signaling pathway activation in microglia and other immune cells, contributing to their response to homeostatic disruption. The expression of CX3CR1 and other microglial markers can also vary across different brain regions, reflecting regional heterogeneity in microglial function.

Figure 4. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CX3CR1 antibody [EPR24267-2] (ab308613).

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F4/80

F4/80 is a 160 KDa glycoprotein found on the surface of macrophages and resting microglia. It is also expressed by other immune and glial cells, but its expression pattern helps distinguish microglia from other glial cell types in the CNS. During neuroinflammatory conditions, disease-associated microglia and microglial cell activation can show altered F4/80 expression, reflecting their transition from a homeostatic to an activated state.

Figure 5. Multiplex immunohistochemistry - Anti-F4/80 antibody [EPR26545-166] (ab300421).

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CD68

CD68 is a lysosomal protein expressed in high levels by macrophages and activated microglia and in low levels by resting microglia. It is one of the key activated microglia protein markers used to identify activated microglia in both research and clinical studies. CD68+ microglia are often associated with the production of inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) and IL-1β. These cytokines contribute to the inflammatory response and can promote immune cell proliferation during neuroinflammation.

Figure 6. Multiplex immunohistochemistry - Anti-CD68 antibody [EPR23917-164] (ab283654).

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CD40

CD40 is a cell surface molecule necessary for antigen presentation and is expressed by activated microglia and macrophages. In the central nervous system, microglia function as antigen-presenting cells by expressing major histocompatibility complex II (MHC II) and co-stimulatory molecules such as CD80 and CD86, activating other immune cells and coordinating the immune response. Microglial antigen presentation is particularly relevant in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, as well as amyotrophic lateral sclerosis, where microglial activation and neuroinflammation play key roles in disease progression.

Figure 7. Flow Cytometry - Anti-CD40 antibody [EPR20735] (ab224639).

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References

1. Bennett, M. L.  et al.  New tools for studying microglia in the mouse and human CNS.  Proc. Natl Acad. Sci. USA  113, 1738–1746 (2016).

2. Schwabenland, M.  et al.  Analyzing microglial phenotypes across neuropathologies: a practical guide.  Acta Neuropathol.  141, 1–22 (2021).

3. Becher, B. & Antel, J. P. Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia.  Glia  17, 1–10 (1996).

4. Ford, A. L., Goodsall, A. L., Hickey, W. F. & Sedgwick, J. D. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.  J. Immunol.  154, 4309–4321 (1995).

5. Schwabenland, M.  et al.  Analyzing microglial phenotypes across neuropathologies: a practical guide.  Acta Neuropathol.  141, 1–22 (2021). (Duplicate of #2)

6. Bhasin, M., Wu, M. & Tsirka, S. E. Modulation of microglial/macrophage activation by macrophage inhibitory factor (TKP) or tuftsin (TKPR) attenuates the disease course of experimental autoimmune encephalomyelitis.  BMC Immunol.  8, 10 (2007).

7. Patel, A. R., Ritzel, R., McCullough, L. D. & Liu, F. Microglia and ischemic stroke: a double-edged sword.  Int. J. Physiol. Pathophysiol. Pharmacol.  5, 73–90 (2013).

8. Ito, D.  et al.  Enhanced expression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain.  Stroke  32, 1208–1215 (2001).

9. Ponomarev, E. D., Shriver, L. P. & Dittel, B. N. CD40 expression by microglial cells is required for their completion of a two-step activation process during central nervous system autoimmune inflammation.  J. Immunol.  176, 1402–1410 (2006).