Neuroscience or neurobiology is the study of the nervous system. This area of biology studies the structure and function of the nervous system at the molecular, developmental, and physiological level. Neurobiology has long been considered one of the most challenging areas of study to fully comprehend. The nervous system is very complex with organs that are hard to reach physically and components such as emotions and consciousness are difficult to measure. However, scientific advances in recent decades have given us better insight to the functions of the nervous system, how these functions are regulated, what causes developmental defects or diseases, and how they can be managed or cured. Research tools, such as antibodies, can help examine these aspects at a molecular level, where proteins can be identified, and their functions can be derived.
Application of antibodies in neurobiology research
Antibodies are used in a variety of applications in neurobiology research. Each application aids in the study of the structure and function of specialized cells within the nervous system. These applications include:
- Detection in western blotting
- Visualization in live and fixed cells or tissues using immunohistochemistry, immunocytochemistry, or immunofluorescence
- Protein enrichment/the study of protein interactions using immunoprecipitation
- Quantitate, characterize, identify, and isolate cell types/populations using flow cytometry and FACS
Antibodies are used in neurobiology to study neuronal markers, cellular differentiation, and disease markers associated with the nervous system. Target-specific antibodies can help detect low-abundance proteins in complex cellular systems such as neurons. The following sections discuss in further detail how antibodies are used as neuronal cell markers, in in vitro differentiation, and as biomarkers for neurodegeneration.
Antibodies as neuronal cell markers
- Glial cells or neuroglia are non-neuronal cells that function to hold neurons in place, supply nutrients to neurons, and defend neurons against pathogens.
- Astrocytes, which are a sub-type of glial cells, are star-shaped cells that support the blood-brain barrier, as well as, provide structural and metabolic support to neurons.
- Oligodendrocytes, another type of glial cell, provide insulation to neurons in the form of myelin.
- Neuroectoderm, which consists of cells from the ectoderm, are important in the development of the nervous system.
- Microglia, a type of glial cell, that function as macrophage cells and are the first form of immune defense in the central nervous system.
Each cell type also expresses specific protein marker profiles that can be used to identify and characterize the cells. This allows researchers to use antibodies, that target these specific protein markers, as neuronal cell markers. For example, in the data below, antibodies specific to GFAP (an astrocyte marker) and NeuN (a mature neuron marker) are used in immunofluorescence to identify localization of these two cell markers. GFAP is found in the cytoskeleton and NeuN is found in the nucleus. In addition to immunofluorescence data, there is western blot data demonstrating that these antibodies are specific to their targets.
Immunofluorescence analysis of GFAP. Immunofluorescence was performed using 70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with GFAP Polyclonal Antibody (Cat. No. PA5-16291) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature, then labeled with Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034) at a dilution of 1:2,000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
Antibody specificity demonstrated for GFAP antibody. Specificity was demonstrated by detection of differential basal expression of the target across tissue tested owing to their inherent genetic constitution. Relative expression of GFAP was observed in mouse brain and rat brain in comparison to mouse liver and rat liver using GFAP Polyclonal Antibody (Cat. No. PA5-16291) in western blot.
Immunofluorescence analysis of NeuN. Immunofluorescence was performed on fixed and permeabilized SH-SY5Y cells for detection of endogenous NeuN using NeuN Recombinant Rabbit Monoclonal Antibody (14H6L24) (Cat. No. 702022, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034, 1:2,000). Panel a) shows representative cells that were stained for detection and localization of NeuN (green), Panel b) is stained for nuclei (blue) using SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Cat. No. R415, 1:300). Panel d) is a composite image of Panels a, b, and c clearly demonstrating nuclear localization of NeuN. Panel e) represents control cells with no primary antibody to assess background.
Antibody specificity demonstrated for NeuN antibody. Specificity was demonstrated by detection of differential basal expression of the target across cells owing to their inherent genetic constitution. Relative expression of NeuN was observed in mouse and rat brain and cerebellum and not mouse and rat heart and kidney tissues using NeuN Recombinant Rabbit Monoclonal Antibody (Cat. No. 702022) in western blot.
Further markers of interest against commonly studied neuronal cell types are listed in the table below.
|Neuronal cell type||Markers of interest|
|beta-3 Tubulin (TUBB3)|
|Glial progenitor cell markers||CD44|
|Presynaptic and post-synaptic markers||Synaptophysin|
|Oligodendrocyte progenitor cell and oligodendrocyte markers||OLIG1|
|Astrocyte markers||Glutamine synthetase|
Antibodies to characterize in vitro differentiation
Access to appropriate samples in neuroscience research can be challenging. Samples can include animal models and primary neurons, which can be difficult to collect, preserve, and process for sample preparation. Cell repositories carry few immortalized neuronal cell lines and the ones that are available are transformed cells obtained from resected tumors. One way to overcome this challenge is by generating specialized cell/organoid structures in the lab using stem cells. Learn more about using in vitro differentiation models to study neuronal protein function and the critical role antibodies play in characterizing these populations in the Behind the Bench Blog: DIY Neurons for Antibody Validation.
Stem cells from various sources can be differentiated into several neuronal lineages to study developmental and disease physiology. For example, Neural Stem Cells (NSCs) that are positive for proteins such as SOX2 and Nestin can be obtained from Embryonic Stem Cell (ESC) differentiation. The NSCs can further be differentiated into mature neurons that express proteins including beta-III tubulin and MAP2. Similarly, induced Pluripotent Stem Cells (iPSCs) can be differentiated into specialized cell types like Retinal Ganglion Cells (RGCs), a neuronal cell type located in the retina, that produce Optineurin, a Golgi-associated retinal protein.
Below, antibodies against four important markers used to characterize cells during cellular differentiation experiments are showcased. SOX1 is a transcription factor that promotes neuronal cell date determination and differentiation. SOX2 is a protein that is required for stem cell maintenance in the central nervous system. PAX3 is known to regulate neurogenesis during development of the peripheral nervous system. And finally, TUBB3 is an early marker of neuronal differentiation.
Western blot analysis of SOX1. Western blot was performed using SOX1 Recombinant Rabbit Monoclonal Antibody (JJ20-40) (Cat. No. MA5-32447) and a 50 kDa band corresponding to SOX1 was observed in Neural Stem Cells, Embryoid Bodies, and Mouse Post-natal Brain
(Day 1), but not in iPSC and Mouse Adult Brain which are reported to be negative. Whole cell lysate (30 ug lysate) of iPSC (Lane 1), Neural Stem Cells (Lane 2), Embryoid Bodies (Lane 3), Mouse Post-Natal Brain (Day 1) (Lane 4) and Mouse Adult Brain (Lane 5) were electrophoresed using NuPAGE 4-12% Bis-Tris gel (Cat. No. NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane by an iBlot 2 Gel Transfer Device
(Cat. No. IB21001). The blots were probed with the primary antibody (1:500 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036, 1:4,000 dilution) using the iBright FL 1000. Chemiluminescent detection was performed using Novex ECL Chemiluminescent Substrate Reagent Kit (Cat. No. WP20005).
Immunofluorescence analysis of SOX2. IF was performed using 70% confluent log phase NTERA-2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with SOX2 Monoclonal Antibody (Btjce), Alexa Fluor 488 (Cat. No. 53-9811-80) at 5 µg/ml in 0.1% BSA, incubated at 4-degree Celsius overnight (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows SOX2 negative cell line HeLa with no signal. Panel f represents control cells with isotype control to assess background. The images were captured at 60X magnification
Western blot analysis of PAX3. Western blot was performed by loading 20 µg of U-87 MG wild type (Lane 1), U-87 MG Cas9 control (Lane 2), and U-87 MG PAX3 knockout (Lane 3) whole cell extracts. The blot was probed with PAX3 Monoclonal Antibody (GT1210) (Cat. No. MA5-31583) (1:2,000 dilution) and Goat anti-Mouse IgG (H+L), Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A28177) (1:4,000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to PAX3. Observed uncharacteristic non-specific band at ~110 kDa.
Immunofluorescence analysis of beta-3 Tubulin. Immunofluorescence analysis was performed on wild type U-87 MG cells (panel a,d), U-87 MG Cas9 cells (panels b,e), and U-87 MG beta-3 Tubulin KO cells (panel c,f). Knockout of beta-3 Tubulin (TUBB3) was achieved by CRISPR-Cas9 genome editing. Cells were fixed, permeabilized, and labelled with beta-3 Tubulin Monoclonal Antibody (2G10-TB3), Alexa Fluor 488 (Cat. No. 53-4510-82) (10 µg/mL). Nuclei (blue) were stained using ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962), and Rhodamine Phalloidin (Cat. No. R415) (1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal (panel c,f) upon CRISPR mediated knockout (KO) confirms that the antibody is specific to beta-3 Tubulin (green). The images were captured at 60X magnification.
Further markers of interest used to characterize different neuronal lineages are listed in the table below.
Antibodies as biomarkers of neurodegeneration
The nervous system is vulnerable to several disorders that can be brought about by injury, tumors, infarcts, autoimmune conditions, degeneration, and structural/developmental defects. Infectious diseases, such as encephalitis and meningitis, can also affect the brain.
There is currently an urgent need to understand neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington’s disease at a cellular and molecular level. This level of understanding could reveal target proteins impacted by these conditions. However, many neurodegenerative diseases share common pathogenic mechanisms and can make the study of the proteins involved even more challenging.
For example, defects in axonal transport has been implicated in both Parkinson’s and Alzheimer’s disease. The SNARE family of proteins that include SNAP25 (synaptosomal-associated protein, molecular mass of 25 kDa) and VAMP1 (vesicle-associated membrane protein-1) play critical roles in facilitating vesicular transport of neurotransmitters across synapses and are found to be mutated in both neurodegenerative disorders.
Another example, where diseases share a pathogenic mechanism, is Amyotrophic Lateral Sclerosis (ALS) and in Spinal Muscular Atrophy (SMA). Abnormal assembly of neurofilaments is found in both diseases. Neurofilament proteins are found in neuron cytoplasm and are important for signal transmissions across axons. Degeneration of neurons in disease conditions releases neurofilaments into the blood or cerebrospinal fluid where they can be detected to aid diagnosis.
To tease apart common disease mechanisms, antibodies can be used in a variety of applications to provide insights into protein signaling pathways and identifying the difference in the structures and functions between normal and diseased conditions. Given the complexity, low-abundance, and often overlapping nature of proteins involved in neuronal disorders, it is imperative that scientists choose antibodies that are certain to recognize their intended targets. Below are examples of data using antibodies tested for specificity against four proteins important to some neurodegenerative diseases.
Western blot analysis of HTT. Western blot was performed by loading 30 µg of SH-SY5Y wildtype control (Lane 1), SH-SY5Y Cas9 control (Lane 2), and SH-SY5Y HTT knockout (Lane 3) whole cell extracts. The blot was probed with Huntingtin Polyclonal Antibody (Cat. No. PA5-85721) using 1:1,000 dilution and Goat anti-Rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036) using 1:4,000 dilution. Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to HTT.
Immunofluorescence analysis of beta Amyloid. IF was performed using 70% confluent log phase PC-3 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with beta Amyloid Monoclonal Antibody (2C8) (Cat. No. MA1-25493) at 1:200 dilution in 0.1% BSA, incubated at 4 degrees Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Cat. No. A32723), at 1:3,000 dilution for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300 dilution). Panel d represents the merged image showing cytoplasm, plasma membrane and nuclear localization. Panel e represents merged image for K-562 cells showing no staining for APP. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Further proteins of interest used as biomarkers of some neurodegenerative diseases are listed in the table below.
|Cell type||Markers of interest|
|Parkinson's disease||alpha Synuclein|
|Amyloid precursor protein|
|Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA)||NEFM|
The nervous system is a challenging area of biology. Research in the neuroscience field helps us understand the intricacies behind the processes, the developmental cues, and the identification of treatment modalities for different neurological diseases. The scope of neuroscience in recent decades has widened significantly to include cellular and molecular aspects of the nervous system and has led to advancements in research models and tools. Neuroscientists are increasingly curious to find ways to predict, prevent, and treat anomalies. However, a lack of quality research reagents poses challenges. Tools such as specificity-verified antibodies can help overcome these challenges and allow researchers to unveil the curiosities surrounding the nervous system.
For Research Use Only. Not for use in diagnostic procedures.