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Table 2 Astrocytic markers and stains

From: Astrocytes reassessment - an evolving concept part one: embryology, biology, morphology and reactivity

Procedures Characteristics Results Observation
Hematoxylin and eosin stain (H-E) [23] Routine staining for basic morphology Nuclear details • Astrocytes are difficult to identify (nuclei: small, pale, ovoidal, euchromatic and centrally situated, are mimicking those of small neurons; cytoplasm and cellular processes are undifferentiated from those of neighbouring neurons)
  Cytoplasm extracellular protein components • The occasionally pericellular hallo (autolitic modification) impose a differential diagnosis with the oligodendrocytes [23]
Mallory’s (phosphotungstic acid – hematoxylin) stain [24] Special stain Astrocyte processes (deep blue)  
Orange-acridine stain [24] Special stain Cellular body • Reveals the astrocytic hyperplasia, without the modification of the cytoplasm aspects [24]
Metallic impregnations [23]   Nuclei • Reveals the cellular characteristic star- shaped aspect
Del Rio Hortega method • Special technique with ammonia silver carbonate Cytoplasm processes • The abundant cytoplasm surrounding the nuclei differentiates the astrocytes from oligodendrocyte
Ramon y Cajal method (see Figures 1 and 2) • Special technique with gold chloride   • The fibrillar aspect of the cytoplasm is due to the material formed by the aggregation of GFAP intermediate filaments
Golgi stain • Special technique with silver nitrate   • The vascular endfeet are easy to identify.
• Protoplasmic astrocytes, due to their proximity to the blood vessels, are able to contact the vessel directly by their cell body
    • The perivascular hallo is considered to be an artefact [23].
Electron microscopy [24]   Cytoplasm intermediate GFAP • Cytoplasm pale , with lack of organelles
• The clear, perivascular spaces indicate excessive dilatation of astrocytic processes due to water imbibitions
• The ultrastructural resemblance between normal and well differentiated neoplastic astrocytes is one of the arguments against the use of this method for positive diagnosis of low grade glioma [24]
Immunohistochemistry    GFAP represents an integrator of the cellular space, but it is also implicated in complex cellular events, such as cytoskeleton reorganisation, myelination, cellular adhesion and several signalling pathways [23, 24].
GFAP (intracytoplasmic protein, with 50 Kda molecular weight, considered the major component of glial fibrils and a marker of astrocytic differentiation) [23, 24] (see Figure 3) • Golden standard for the definition of astrocytes Cell body • Fibrillary astrocytes contain a massive amount of GFAP in their cell bodies and processes unlike protoplasmic astrocyte.
• There are different clones of antiGFAP antibodie, characteristic to the different research Cell processes (positive immunostaining reaction: brown spots) • Protoplasmic astrocytes are much larger than their GFAP-defined profiles due to the presence of numerous fine processes that are GFAP-negative
• Laboratories (e.g. GF2 DAKO clone; Astro 1) [23, 24]   • In astrocytomas, along with the enhancement of malignity, the intracellular quantity of GFAP is progressively reduced; therefore the evaluation of GFAP immunohistochemical staining will enable the immunophenotypic characterisation of the investigated glial tumors and the confirmation of histopathological diagnosis
• Not all the cells in the CNS that express GFAP are astrocytes (e.g: astrocyte-like cells from the SVZ-derived from radial glia, ependymal cells) [1, 25, 26]
• GFAP has also been located in rat kidney glomeruli and peritubular fibroblasts [1, 27], Leydig cells of the testis [1, 28], skin keratinocytes [1, 29], osteocytes of bones, chondrocytes of epiglottis, bronchus [1, 30], and stellate-shaped cells of the pancreas and liver [1]
S100B (belongs to the S100 family of EF-band calcium binding proteins [1, 31]). There are different clones of anti S100 antibodies, characteristic to the different research laboratories (e.g. MAB079, CBL410.) Cell membrane • Expressed by a subtype of mature astrocytes that ensheath blood vessels and by NG2-expressing astrocytes [1, 31]
Other astrocytic markers    
GLT-1 (the glutamate transporters GLAST) [6]    • GLT-1 is expressed by all astrocytes and provide punctuate staining [6]
Human EAAT2 (excitatory amino acids, 1 and 2 for human brain) [6]    
Glycogen granules [6]   Cytoplasm  
Gglutamine synthase (GS) [1, 3235] GS- enzyme that catalyzes the conversion of ammonia and glutamate to glutamine Cytoplasm GS is expressed also by oligodendrocytes [1, 3235]
Kir4.1 (inwardly rectifying K+ channels) [1, 36, 37]    Kir4.1 are only expressed by a subset of astrocytes [37]
Aquaporin 4 channels [1, 38]   Cell processes Aquaporin 4 channels is localized in some parts of the astrocytic processes rendering identification of the whole cell difficult to interpret [38]
AldhL1 (aldehyde dehydrogenase 1 family, member L1) [1, 39].   Genome • All astrocytes
Battery of tests [40]• GFAP-driven GFP (green fluorescent protein) expressionGFAPprotein expression, S100ß immunostaining Combinatorial approach   • Nine different classes of astrocytes has been identified, that included Bergmann glia, ependymal glia, fibrous astrocytes, marginal glia, perivascular glia, protoplasmic astrocytes, radial glia, tanycytes and velate glia [3, 40]
• GFAP expression glutamate response [41] • Define the phenotype of an astrocyte population as (GFAP+/NG2-; T+/R-) which is distinct from NG2-glia (GFAP-/NG2+ T-/R+) [41]
Dye-filling techniques [6, 42](e.g. sharp electrode, patch clamp recordings, single cell electroporation) Special techniques that identify cells recorded in situ after filling them with a dye present in a micro-electrode Cell body • This technique has the advantage that the cells to be studied can be preselected in living tissue [6, 42]
It is suplemented by use of presumed astrocyte- Cell processes • However, proteins and promoter activation are subjects to change. Hence one can have a GFAP(-) cell that one should call an astrocyte because it has these other properties [6, 42]
Specific promoters to drive synthesis of fluorescent proteins   • Using these procedures the domain organisation of astrocytes has been demonstrated along with the fusiform morphology of astrocyte nucleus, both playing a possible role in pathology [3, 43, 44]
Transgenic techniques (use transgenic mice) [1] Visualize fluorescent astrocytes Cell body • Mice specific for astrocytes express [1]
Cell processes - GFP
- Enhanced GFP under the human GFAP promoter (hGFAP-GFP mice)
- GLT-1-GFP
- BLBP-dsRed2