Revision 1

#38554Store at -20C

1 Kit

(6 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Doublecortin (E6O6A) Rabbit mAb 91954 20 µl 45 kDa Rabbit IgG
NCAM1 (CD56) (E7X9M) XP® Rabbit mAb 99746 20 µl 120 to 220 kDa Rabbit IgG
NeuroD1 (D90G12) Rabbit mAb 7019 20 µl 49 kDa Rabbit IgG
β3-Tubulin (D71G9) XP® Rabbit mAb 5568 20 µl 55 kDa Rabbit IgG
TBR1 (D6C6X) Rabbit mAb 49661 20 µl 74 kDa Rabbit IgG
Stathmin (D1Y5A) Rabbit mAb 13655 20 µl 19 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Immature Neuron Marker Antibody Sampler Kit provides an economical means for detecting immature neuron proteins by western blot. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

The antibodies in this kit serve to characterize and identify immature neurons. During development, radial glia (RG) cells located in the ventricular zone (VZ) of the brain divide asymmetrically, each producing a neuronal and RG daughter cell. The daughter RG cell is also known as a neural progenitor cell (NPC) or an intermediate progenitor cell (IPC). Newly formed IPCs migrate to the subventricular zone (SVZ) where they divide symmetrically, each giving rise to two post-mitotic neurons that can then migrate to their final destination. In adulthood, NPCs reside within the subgranular zone (SGZ) of the dentate gyrus, and the adult SVZ, which surrounds the lateral ventricles of the cerebral cortex. NPCs within the SGZ and SVZ divide and give rise to immature neurons (1). The cytoskeleton of these cells plays an important role in generating neuronal processes. The cytoskeleton consists of three types of cytosolic fibers: actin microfilaments, intermediate filaments, and microtubules. β3-tubulin is one of six β-tubulin isoforms that make up the building blocks of microtubules (2). Stathmin is a tubulin binding protein that regulates microtubule dynamics in a phosphorylation dependent manner. Stathmin is heavily expressed during neuronal development, mediating differentiation and synaptic plasticity (3,4). Doublecortin is a microtubule-associated protein that facilitates neurite outgrowth and cell migration (5). The dual expression of doublecortin and NCAM (neural cell adhesion molecule, CD56), combined with the lack of expression of mature neuronal markers, is evidence of an immature neuronal phenotype (6). NCAM mediates neuronal attachment, neurite extension, and cell to cell interactions through homo and heterophilic interactions. Polysialic acid (PSA) post-translational modification of NCAM disrupts cell to cell adhesion, promoting axonal growth, cell migration, and synaptic plasticity during neurogenesis (7-9).

Transcription factors also play a key role in immature neuron growth and differentiation. NeuroD1 is a member of the basic helix-loop-helix (bHLH) family of transcription factors. These proteins function by forming heterodimers with E-proteins and binding to the canonical E-box sequence CANNTG (10,11). Neuronal activity results in CaMKII-mediated phosphorylation of NeuroD1 at Ser336, which is necessary for the formation and growth of dendrites (12,13). T-box, brain, 1 (TBR1) is a transcription factor important in vertebrate embryo development. As a member of the T-Box family of transcription factors, TBR1 is expressed in postmitotic glutamatergic projection neurons (14). During cortical neurogenesis, sequential expression of transcription factors Pax6, TBR2, and TBR1 regulates discrete steps in projection neuron differentiation (15).

  1. Martínez-Cerdeño, V. and Noctor, S.C. (2018) Front Neuroanat 12, 104.
  2. Jiang, Y.Q. and Oblinger, M.M. (1992) J Cell Sci 103 (Pt 3), 643-51.
  3. Chauvin, S. and Sobel, A. (2015) Prog Neurobiol 126, 1-18.
  4. Uchida, S. et al. (2014) Nat Commun 5, 4389.
  5. Reiner, O. et al. (2004) Cell Cycle 3, 747-51.
  6. Coviello, S. et al. (2022) Front Neuroanat 16, 851432.
  7. Seidenfaden, R. et al. (2003) Mol Cell Biol 23, 5908-18.
  8. Bonfanti, L. and Seki, T. (2021) Cells 10, 2542.
  9. Wędzony, K. et al. (2013) Pharmacol Rep 65, 1471-8.
  10. Schonhoff, S.E. et al. (2004) Endocrinology 145, 2639-44.
  11. Sharma, A. et al. (1999) Mol Cell Biol 19, 704-13.
  12. Chae, J.H. et al. (2004) Mol Cells 18, 271-88.
  13. Gaudillière, B. et al. (2004) Neuron 41, 229-41.
  14. Hevner, R.F. et al. (2001) Neuron 29, 353-66.
  15. Englund, C. et al. (2005) J Neurosci 25, 247-51.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

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    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #38554

    Immature Neuron Marker Antibody Sampler Kit

    Immature Neuron Marker Antibody Sampler Kit: Image 1 Expand Image
    Confocal immunofluorescent analysis of WERI-Rb-1 (left, positive) and KARPAS 299 (right, negative) using NeuroD1 (D90G12) Rabbit mAb (green) and DAPI #4083 (blue).
    Immature Neuron Marker Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from various cell lines using Stathmin (D1Y5A) Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 3 Expand Image
    Immunohistochemical analysis of paraffin-embedded human brain using TBR1 (D6C6X) Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of HeLa and human cerebellum using β3-Tubulin (D71G9) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 5 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Immature Neuron Marker Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various tissues and cell lines using Doublecortin (E6O6A) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
    Immature Neuron Marker Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines and tissues using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    Immature Neuron Marker Antibody Sampler Kit: Image 8 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Stathmin (D1Y5A) Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 9 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse brain using TBR1 (D6C6X) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Immature Neuron Marker Antibody Sampler Kit: Image 10 Expand Image
    Confocal immunofluorescent analysis of mouse cerebellum using β3-Tubulin (D71G9) XP® Rabbit mAb (green) and Tau (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Immature Neuron Marker Antibody Sampler Kit: Image 11 Expand Image
    Immunoprecipitation of Doublecortin from mouse neonatal brain tissue extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Doublecortin (E6O6A) Rabbit mAb. Western blot analysis was performed using Doublecortin (E6O6A) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
    Immature Neuron Marker Antibody Sampler Kit: Image 12 Expand Image
    Immunoprecipitation of NCAM1 protein from SH-SY5Y cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is NCAM1 (CD56) (E7X9M) XP® Rabbit mAb. Western blot analysis was performed using NCAM1 (CD56) (123C3) Mouse mAb #3576. Anti-mouse IgG, HRP-linked Antibody #7076 was used as the secondary antibody.
    Immature Neuron Marker Antibody Sampler Kit: Image 13 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse kidney using Stathmin (D1Y5A) Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded E13 mouse embryo using TBR1 (D6C6X) Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 15 Expand Image
    Confocal immunofluorescent analysis of P19 cells that were differentiated with retinoic acid, using β3-Tubulin (D71G9) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5 #4084® (fluorescent DNA dye).
    Immature Neuron Marker Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of extracts from various human cell lines using NeuroD1 (D90G12) Rabbit mAb (upper), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Immature Neuron Marker Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma with staining of peripheral nerve using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb performed on the Leica® BOND Rx.
    Immature Neuron Marker Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Stathmin (D1Y5A) Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis of paraffin-embedded rat brain, cortex (left) and cerebellum (right), using TBR1 (D6C6X) Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lymph node using Stathmin (D1Y5A) Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 22 Expand Image
    Confocal immunofluorescent analysis of E14.5 mouse cortex using TBR1 (D6C6X) Rabbit mAb (green) and GFAP (GA5) Mouse mAb (Alexa Fluor® 555 Conjugate) #3656 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Immature Neuron Marker Antibody Sampler Kit: Image 23 Expand Image
    Immunoprecipitation of β3-Tubulin from mouse brain tissue extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is β3-Tubulin (D71G9) XP® Rabbit mAb. Western blot analysis was performed using β3-Tubulin (D71G9) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as the secondary antibody.
    Immature Neuron Marker Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded human renal cell carcinoma using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 25 Expand Image
    Confocal immunofluorescent analysis of C2C12 cells using Stathmin (D1Y5A) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Immature Neuron Marker Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human gastrointestinal stromal tumor using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal rhesus monkey spleen using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 29 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse ovary using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 30 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse prostate using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse colon using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded endometrioid adenocarcinoma using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb (left) or NCAM (CD56) (123C3) Mouse mAb #3576 (right). These two antibodies detect independent, unique epitopes on human NCAM1. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
    Immature Neuron Marker Antibody Sampler Kit: Image 33 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
    Immature Neuron Marker Antibody Sampler Kit: Image 34 Expand Image
    Immunohistochemical analysis of paraffin-embedded SH-SY5Y cell pellet (left, positive) or MCF7 cell pellet (right, negative) using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb.
    Immature Neuron Marker Antibody Sampler Kit: Image 35 Expand Image
    Confocal immunofluorescent analysis of mouse spinal cord (left), or rat brainstem (right) using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb (green) and S6 Ribosomal Protein (54D2) Mouse mAb (Alexa Fluor® 647 Conjugate) #5548 (red). Tissue was mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Immature Neuron Marker Antibody Sampler Kit: Image 36 Expand Image
    Confocal immunofluorescent analysis of SH-SY5Y cells (left, positive) or HeLa cells (right, negative) using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb (green) and S6 Ribosomal Protein (54D2) Mouse mAb (Alexa Fluor® 647 Conjugate) #5548 (red). Cells were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Immature Neuron Marker Antibody Sampler Kit: Image 37 Expand Image
    Confocal immunofluorescent analysis of Neuro-2a cells (left, high-expressing) or C2C12 cells (right, low-expressing) using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb (green) and S6 Ribosomal Protein (54D2) Mouse mAb (Alexa Fluor® 647 Conjugate) #5548 (red). Cells were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Immature Neuron Marker Antibody Sampler Kit: Image 38 Expand Image
    Flow cytometric analysis of HeLa cells (blue) and SH-SY5Y cells (green) using NCAM1 (CD56) (E7X9M) XP® Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.