Neuroscience Cell Identity Markers

Neurons Astrocytes Oligodendrocytes Microglia

The nervous system consists of a complex variety of neurons and non-neuronal cells. The neuronal compartment alone contains billions of neurons, with a not yet fully characterized number of neuronal subtypes.

Major types of brain cells

Neurons

Neurons are electrically excitable cells that transmit signals (electrical and chemical) supporting a wide range of functions including cognition, sensory perception and movement. Morphologically, neurons contain four well defined structural compartments including dendrites, soma, axon and synaptic terminal. Morphology and connectivity have traditionally been instrumental in neuronal identification. Nevertheless, depending on investigator’s objectives, neuronal identification often requires the detection of several molecular markers to:

  1. Distinguish neurons from other brain cells (e.g., neurons vs glial)
  2. Determine neuronal identity (e.g., subtypes)
  3. Define a neuron’s function (e.g., excitatory vs inhibitory)
  4. Establish synaptic partners (e.g., pre- and post-synaptic)

Commonly Used Neuronal Markers

Immature Neuron Markers Mature Neuron Markers Functional Neuron Markers Synaptic Neuron Markers

Doublecortin, NCAM, NeuroD1

Enolase 2/NSE, NeuN, MAP2*, beta-III Tubulin, Neurofilament Light, Neurofilament Medium, Neurofilament Heavy, GAP-43

ChAT, Tyrosine Hydroxylase, GAD65/GAD67, VGLUT1, VGLUT2, Pet1, SERT

PSD-95, Synaptophysin, Bassoon

Notes: *In comparison with beta-III Tubulin, MAP2 labels more mature neurons.



NCAM: Immature Neuron Marker

 

GAP-43: Mature Neuron Marker

Neural Cell Adhesion Molecule 1 (NCAM-1)/CD56 serves as an immature neuronal marker.

  

Growth-associated protein (GAP), GAP-43 serves as a mature neuronal marker.

(NCAM-1)/CD56 was detected in SH-SY5Y cells with Goat Anti-Human/Mouse NCAM-1/CD56 Polyclonal Antibody (AF2408). (red) Cells were stained with NorthernLights 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (NL001). Phalloidin (green) stained Actin filaments and DAPI (blue) stained the cell nuclei. NCAM-1/CD56 expression was localized to the plasma membrane.

  

GAP-43 was detected in a mature retinal ganglion cell with Sheep Polyclonal GAP-43 antibody [NBP1-41123]. Staining shows axon outgrowth.


Tyrosine Hydroxylase: Functional Neuron Marker

 

Synaptophysin: Synaptic Neuron Marker

Tyrosine Hydroxylase (TH), serves as a functional neuronal marker and helps identify dopaminergic neurons.

  

Synaptophysin serves as a synaptic neuronal marker and helps identify presynaptic terminals.

Tyrosine Hydroxylase Antibody was detected in FFPE tissue section of human brain /substantia nigra with Mouse Monoclonal Tyrosine Hydroxylase antibody (5C7.2E8) [NBP2-42211] used at 15 ug/ml. Tissue shows strong immunostaining of Tyrosine Hydroxylase positive neurons.

  

Synaptophysin was detected in perfusion fixed frozen sections of mouse brain hippocampus with Mouse Anti-Human Synaptophysin Monoclonal Antibody (MAB5555) used at 15 µg/mL. (red) Tissue was stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (Catalog NL007). Nuclei were counterstained with DAPI (blue). Synaptophysin staining is present in cytoplasm and nuclei.

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Glia

Glial cells are non-neuronal cells found in the central and peripheral nervous system including microglia and macroglia cells. Among these, macroglia cells include astrocytes, oligodendrocytes, ependymal cells, radial glia, Schwann cells and satellite cells. Glial cells function to support a variety of neuronal activities including migration, axonal outgrowth and synaptic activity. Additionally, glial cells provide neurons with trophic and metabolic support and help maintain synaptic homeostasis.


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Protoplasmic and fibrous astrocytes

Astrocytes are the most ubiquitous and diverse of the glial cells, being both functionally and molecularly diverse. Functionally, astrocytes play a role in neurotransmitter clearance, ion homeostasis and in the regulation of synapse number, thereby modulating neuronal activity through different mechanisms. Their molecular diversity is exemplified by the variable expression of key proteins including glial fibrillar acidic protein (GFAP), glutamate transporter (Glt1/EAAT2) and the gap junction protein connexin 30 (Cx30). Particularly, GFAP expression, often used as a reliable astrocyte marker, is not always expressed by astrocytes and is more common to reactive and white matter astrocytes. Markers for mature astrocytes include aldehyde dehydrogenase family 1 member L1 (Aldh1L1), aldolase C (AldoC), glutamate transporter-1 (Glt1), S100 calcium-binding protein B (S100b) and Aquaporin 4.

Two types of astrocytes are currently recognized, although morphological studies suggest a greater diversity.


Protoplasmic Fibrous
S100b + GFAP +
Gray matter White matter

Glial Fibrillary Acidic Protein (GFAP) is an astrocyte marker which may be detected by immunostaining astrocytes in culture and within brain tissue.

    

S100 calcium-binding protein B (S100B) is expressed and secreted by astrocytes and serves as a pan-astrocytic marker and identity marker in brain tissue immunostaining.

GFAP was detected in immersion fixed rat astrocytes using 10 µg/mL Sheep Anti-Human GFAP Antigen Affinity-purified Polyclonal Antibody (AF2594) for 3 hours at room temperature. (red) Cells were stained with the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (NL010) and counterstained with DAPI (blue).

  

S100B was detected in immersion fixed paraffin-embedded sections of human Alzheimer's brain using Goat Anti-Human S100B Antigen Affinity-purified Polyclonal Antibody (AF1820) at 1.7 µg/mL overnight at 4°C. (brown) Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit; (CTS008) and counterstained with hematoxylin (blue).


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Oligodendrocyte

Oligodendrocytes provide metabolic support and myelinate neuronal axons in the CNS. Insulating-myelin sheaths are indispensable for fast action potential conduction along the neuronal axon. Oligodendrocytes may be molecularly identified by the expression of various markers including myelin basic protein (MBP), Sox10, myelin oligodendrocyte glycoprotein (MOG), carbonic anhydrase II, CNPase (2’,3’-cyclic nucleotide 3’-phosphohydrolase), Nogo-A and proteolipid protein (PLP). More recently a new marker for mature oligodendrocytes, Tmem10/Opalin, was identified which is co-expressed with other markers including MOG and MBP in myelinated fibers.


CNPase expression in adult mouse brain IHC

    

Myelin Basic Protein (MBP) is a component of myelin sheath in the CNS and is expressed by oligodendrocyte at late stages of their differentiation, therefore serves to identify oligodendrocyte linage.

CNPase was detected in mouse brain with CNPase Polyclonal Antibody [NB100-1935]. A tissue section through an adult mouse brain showing CNPase (brown staining) in white matter tracts and the granule cell layer of the cerebellum.

  

MBP was detected in mouse brain with Myelin Basic Protein Monoclonal Antibody  (2H9) [NBP2-22121]. Mouse brain cryosections were stained with MBP antibody at a dilution of 1:400 and secondary antibody anti-mouse Alexa Fluor 555 IgG. Nuclei were counterstained with DAPI (blue).



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Microglia

Microglia are the immune cells of the nervous system. They are macrophages that serve as the first line of immune defense in the CNS targeting damaged neurons, plaques and infectious agents for phagocytosis. Even in the absence of a trigger or insult to the brain, microglia constantly monitor their surrounding tissues and participate in the maintenance of homeostasis. During development, microglia derived factors influence neuronal survival, either supporting survival or inducing cell death via apoptosis.

Identification of microglia has traditionally relied on markers such as CD45 expression and more recently Cx3cr1. A nuance in the field has been the identification of Tmem119 as a microglia specific marker.



TMEM119 expression in human cerebral cortex IHC

Orthogonal Strategies Validation. Immunohistochemistry-Paraffin: TMEM119 Antibody [NBP2-30792] - Staining in human cerebral cortex and liver tissues. Corresponding TMEM119 RNA-seq data are presented for the same tissues. Staining of human cerebral cortex shows moderate to strong membranous positivity in microglia. Staining of human liver shows no positivity in hepatocytes as expected.





P2Y12/P2RY12 expression  in microglia of human cerebral cortex IHC

Immunohistochemistry-Paraffin: P2Y12/P2RY12 Antibody [NBP2-33870] - Staining of human cerebral cortex shows strong cytoplasmic positivity in microglia.





Following an insult to the brain, for example by infectious pathogens crossing the blood-brain barrier, microglia transform from a ramified to an amoeboid morphology, which correlate with their inactive and active states, respectively. Upon activation, microglia undergo changes in gene expression that underscore their new functionalities, for example the ability to produce inflammatory substances and phagocytose.



Microglia transform from ramified to amoeboid morphology upon activation



Genes modulated by LPS-induced microglia activation

Up-regulated expression Down-regulated expression
Lcn2 Tmem119
Ccl3 Fcrls
Cxcl10 Olfml3
Ccl5 Ltc4s
IL-1b Adora3
Tlr2 P2ry12

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