Cortical Layer Markers

Learn about the development of cortical layers, useful markers for the different layers and explore antibodies against these proteins to use in your IHC, ICC-IF or Western blot assays.

Cortical layer markers are useful tools for studying the development, functional neuroanatomy and pathology of the cerebral cortex. The cerebral cortex is organized into six layers of both morphologically and functionally divergent neurons. The different layers can be identified using antibodies against protein markers expressed in specific layers.

Development and characteristics of cortical layers

Cortical layers are formed during embryonal development

The neuronal layers in the cortex generate during embryonal development, in a strictly regulated manner. Layers V and VI, the deeper layers neurons,  generate after the formation of the cortical plate. Layers II-IV, the upper layers neurons, are formed after the deeper layers. Glial cells, including astrocytes and oligodendrocytes generate at the latest stages of cortical development.

Tissue expression of selected transcriptional factors involved in the development of cerebral cortex.
Tissue expression of selected transcriptional factors involved in the development of cerebral cortex. Immunohistochemical staining with Anti-LHX2 antibody (AMAb91404) shows strong immunoreactivity in the developing cortex and sensory organs of mouse embryo E11 (A). PAX6 expression is detected in both developing brain and sensory organs (B), including eye (C) (Anti-PAX6 antibody AMAb91372). MEF2C is primarily expressed in the upper layers of the developing cortex, as shown by Anti-MEF2C antibody (AMAb90728) in mouse embryo E14 (D).

The neocortical neurogenesis is dependent on several transcriptional factors. For instance, LHX2 and PAX6 together play a crucial role in the specification of neocortical progenitors which give rise to the projection neurons. MEF2C is another example of a transcription factor essential for normal neural development and spatial distribution in the neocortex. 

Anatomical and functional diversity in neurons of different layers

Neurons of different layers are both anatomically and functionally diverse in the mature neocortex. Neuronal cell morphology, physiological properties, and anatomical connections vary between the layers. Neurons of layers II/III, along with a subset of neurons of layer V, contribute mostly to intracortical connections. This includes callosal projections to the contralateral cerebral hemisphere.  Layer V corticofugal neurons target the midbrain, hindbrain and spinal cord, while layer VI neurons project mainly to the thalamus.

Laminar distribution of protein expression profiles in the cerebral cortex of rat.
Laminar distribution of protein expression profiles in the cerebral cortex of rat. RELN immunoreactivity is mainly detected in the neuropil of layer I (Anti-RELN antibody AMAb91365) (A, F). CUX1 expression is seen in layers II-IV (Anti-CUX1 antibody HPA003277) (B, G). BCL11B (CTIP2) is expressed primarily in layers V and VI (Anti-BCL11B antibody HPA049117) (C, H). Layer VI neurons show strong expression of FOXP2 (Anti-FOXP2 antibody AMAb91362) (D, I). Layer VIb can be identified by presence of CTGF immunoreactivity (Anti-CTGF antibody AMAb91366) (E, J).

The cortical layer markers CUX1, POUF3F2, BCL11B, and FPXP2

Protein expression profiles differ in neurons of various layers. For instance, upper layers neurons can be identified by expression of CUX1 and POU3F2 (BRN2), the neurons of layer V by expression of BCL11B (CTIP2) and neurons of layer VI can be identified by FOXP2 expression. 

Laminar structure of cerebral cortex of rat
Laminar structure of cerebral cortex of rat shown by multiplexed immunofluorescence with the Anti-CUX1 antibody (HPA003277, in blue), the Anti-NECAB1 antibody (AMAb90800, in green) and the Anti-FOXP2 antibody (AMAb91362, in red). Note that CUX1 is mainly expressed in layers II-III, NECAB1 shows strongest expression in layer IV neurons, while FOXP2 is primarily present in layer VI neurons.

Antibodies targeting cortical layer I

Layer 1: Anti-RELN monoclonal antibody (AMAb91365)     

  • validated for IHC
  • verified species reactivity: human, mouse, rat

Layer 1: Anti-RELN polyclonal antibody (HPA046512)          

  • validated for ICC-IF
  • verified species reactivity: human

Antibodies targeting upper cortical layers markers

Antibodies targeting proteins in the cortical layers II, III, IV, and V include CUX1 (CUTL1) and POU3F2 (BRN2).

Layer 2/3: Anti-RASGRF2 polyclonal antibody (HPA018679)

  • IHC
  • human, rat

Layer 2/3: Anti-CALB1 polyclonal antibody (HPA023099)    

  • IHC, WB, ICC-IF
  • human, mouse, rat

Layer 2/3-4: Anti-CUX1 monoclonal antibody (AMAb91352)           

  • IHC, ICC-IF
  • human

Layer 2/3-4: Anti-CUX1 monoclonal antibody (AMAb91353)           

  • IHC, ICC-IF
  • human

Layer 2/3-4: Anti-CUX1 polyclonal antibody (HPA003277)

  • IHC
  • human, mouse, rat

Layer 2/3, 4 and 5b: Anti-POU3F2 (BRN2) polyclonal antibody (HPA056261)         

  • ICC-IF
  • human, mouse, rat

Layer 2/3, 4 and 5b: Anti-POU3F2 (BRN2) monoclonal antibody (AMAb91406)     

  • IHC, WB, ICC-IF
  • mouse

Layer 2/3, 4 and 5b: Anti-POU3F2 (BRN2) monoclonal antibody (AMAb91407)     

  • IHC, ICC-IF
  • mouse, rat

Layer 2-4 (mainly 4): Anti-NECAB1 monoclonal antibody (AMAb90798)     

  • IHC, WB
  • human, mouse, rat

Layer 2-4 (mainly 4): Anti-NECAB1 monoclonal antibody (AMAb90800)     

  • IHC, WB
  • human

Layer 2-4 (mainly 4): Anti-NECAB1 monoclonal antibody (AMAb90801)     

  • IHC, WB
  • human, mouse, rat

Layer 2-4 (mainly 4): Anti-NECAB1 polyclonal antibody (HPA023629)         

  • IHC, WB
  • human, mouse, rat

Layer 2-4 (mainly 4): Anti-NECAB1 polyclonal antibody (HPA031262)         

  • IHC
  • human

Antibodies targeting deeper cortical layers markers

Antibodies targeting deep or low layer, layer V and VI include PCP4 and BCL11B (CTIP2).

Layer 5: Anti-PCP4 polyclonal antibody (HPA005792)          

  • IHC, WB
  • human, mouse, rat

Layer 5: Anti-CNTC6 polyclonal antibody (HPA016645)       

  • IHC
  • human

Layer 5-6: Anti-BCL11B (CTIP2) polyclonal antibody (HPA049117)  

  • IHC, ICC-IF
  • human, mouse, rat

Layer 6: Anti-FOXP2 monoclonal antibody (AMAb91361)   

  • IHC, ICC-IF
  • human, mouse

Layer 6: Anti-FOXP2 monoclonal antibody (AMAb91362)   

  • IHC, ICC-IF
  • human, mouse, rat

Layer 6: Anti-FOXP2 polyclonal antibody (HPA000382)       

  • IHC
  • human, mouse, rat

Layer 6: Anti-TLE4 polyclonal antibody (HPA065357)          

  • WB, ICC-IF
  • mouse, rat

Layer 6b: Anti-CTGF monoclonal antibody (AMAb91366)    

  • IHC, WB
  • human, mouse, rat

Layer 6b: Anti-CTGF polyclonal antibody (HPA031075)        

  • IHC, WB, ICC-IF
  • human