NELL2 Antibody

What is NELL2 and what does it do in neural tissues?

NELL2 (Neural Epidermal Growth Factor-Like Like Protein 2) is predominantly expressed in neural tissues where it regulates neuronal differentiation, polarization, and axon guidance . This neuron-specific glycosylated protein contains multiple functional domains including Ca2+ binding domains, suggesting its multifunctional roles in neural cells . Research demonstrates that NELL2 is critical for neuronal polarization and normal axon growth, with knockdown studies showing deterioration of neuronal development including inhibition of neuronal progression, decreased axon growth, and increased axon branching . NELL2 functions primarily as a secreted protein rather than an integral membrane protein, as confirmed by fractionation experiments and immunostaining of non-permeabilized cells .

How does NELL2 expression change during neuronal development?

NELL2 expression undergoes significant changes during neuronal development, both quantitatively and qualitatively. In cultured rat embryonic hippocampal neurons, endogenous NELL2 expression gradually increases in parallel with the progression of developmental stages . Immunocytochemistry studies show that NELL2 is expressed and distributed throughout neurons at stages 1-3 of development, with mRNA levels increasing as development progresses . The subcellular distribution pattern also changes dramatically during development - in neural stem cells, NELL2 appears as multiple small puncta (16-65 per cell) distributed throughout the cytoplasm, while in differentiated neurons, the number decreases significantly to only 1-5 puncta per cell, but these puncta become significantly larger and elongated . This shift in expression pattern likely reflects NELL2's changing functions during neural development.

What fixation and permeabilization protocols work best for NELL2 immunostaining?

For optimal NELL2 immunostaining, cells should be washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature, then washed again with PBS . Permeabilization should be performed by incubating in PBS containing 0.25% Triton X-100, 5% normal goat serum, and 5% skim milk for 30 minutes at room temperature . This permeabilization step is crucial, as studies have shown that non-permeabilized cells do not show NELL2 immunoreactivity, confirming that NELL2 exists primarily as an intracellular or secreted protein rather than a membrane-bound protein . For primary antibody incubation, anti-NELL2 antibodies are typically used at concentrations around 0.4 μg/ml and incubated overnight at 4°C . For double-labeling experiments with cytoskeletal proteins, antibodies against β-actin (1:500) and α-tubulin (1:1000) have been successfully used alongside NELL2 antibodies .

What controls should be included when using NELL2 antibodies?

When using NELL2 antibodies, several controls are essential to ensure specificity and biological relevance:

• Negative controls: Include samples incubated with secondary antibody only (primary antibody omitted) to assess background staining and autofluorescence.

• siRNA knockdown controls: Compare NELL2 staining in cells transfected with NELL2-specific siRNA versus control siRNA. Studies have shown that siRNA targeting NELL2 can achieve approximately 50% reduction in mRNA levels, resulting in visible changes in staining patterns .

• Expression vector controls: Include cells transfected with NELL2 expression vectors (e.g., NELL2-EGFP or NELL2-FLAG) as positive controls to confirm antibody binding to overexpressed protein .

• Rescue experiments: After NELL2 knockdown, add purified recombinant NELL2 protein to the culture medium to restore NELL2 signaling. This approach has been used to demonstrate that exogenous NELL2 can fully rescue the proliferation inhibition caused by NELL2 silencing in Ewing sarcoma cells .

• Co-localization controls: Include double-labeling with established cell-type markers (e.g., NESTIN for NSCs, NEUN for neurons, CNPase for oligodendrocytes) to confirm cell-type specific expression patterns .

How can I distinguish between secreted and intracellular forms of NELL2?

Distinguishing between secreted and intracellular NELL2 requires specific methodological approaches:

For intracellular NELL2 detection:

• Perform immunocytochemistry with cell permeabilization using 0.25% Triton X-100 to detect intracellular NELL2 .

• Use confocal microscopy to visualize the punctate distribution of NELL2 within different subcellular compartments.

For secreted NELL2 detection:

• Collect conditioned media from cultured cells and concentrate proteins using trichloroacetic acid precipitation .

• Perform Western blotting of the concentrated media using anti-NELL2 antibodies or anti-tag antibodies for tagged NELL2 constructs .

• For binding studies, produce C-terminally tagged NELL2 (e.g., NELL2-FLAG) and incubate target cells with the purified protein, followed by washing and analysis of bound NELL2 by immunoblotting .

For comprehensive analysis, combine these approaches with subcellular fractionation experiments to separate membrane, cytosolic, and nuclear fractions, followed by Western blotting with NELL2 antibodies . Fractionation studies have confirmed that NELL2-EGFP is not localized in membrane fractions, supporting its identity as a secreted rather than integral membrane protein .

How can NELL2 antibodies be used to study neuronal polarization?

NELL2 antibodies provide valuable tools for studying neuronal polarization through several methodological approaches:

• Expression analysis: Use NELL2 antibodies to track the temporal expression of NELL2 during the establishment of neuronal polarity in primary hippocampal neurons at stages 1-3 of development (0-48 hours in culture) . Immunostaining reveals that NELL2 is expressed and distributed throughout neurons during these early polarization stages .

• Functional studies: Combine NELL2 immunostaining with genetic manipulation approaches. Overexpression of NELL2 has been shown to stimulate neuronal polarization and axon growth, while knockdown of NELL2 results in deterioration of neuronal development, including inhibition of neuronal development progression, decreased axon growth and increased axon branching . NELL2 antibodies can be used to confirm expression changes and correlate them with morphological effects.

• Signaling pathway analysis: Studies have shown that inhibitors against extracellular signal-regulated kinase (ERK) dramatically inhibit NELL2-induced progression of neuronal development and axon growth . NELL2 antibodies can be used to determine whether ERK inhibition affects NELL2 expression or localization.

• Receptor interaction studies: Although NELL2 has been identified as a ligand for Robo receptors in some contexts, studies in hippocampal neurons suggest that NELL2 acts on axon development through signaling systems other than Robo2/3 . NELL2 antibodies can be used in co-localization studies to identify potential alternative receptors or binding partners.

What can NELL2 antibodies reveal about oligodendrocyte biology?

The discovery of robust NELL2 expression in oligodendrocytes using NELL2 antibodies represents a significant finding that contradicts previous reports from animal models . In human brain organoids, NELL2 antibody staining revealed that 97.3% of CNPase-positive oligodendrocytes exhibited a particularly high density of NELL2 puncta . This unexpected finding has prompted investigations into the relevance of NELL2 in oligodendrocytes, with AI-based analysis revealing a previously unknown link between NELL2 and white matter diseases .

NELL2 antibodies can be used to:

• Compare NELL2 expression patterns between rodent and human oligodendrocytes to understand species differences in NELL2 function.

• Track NELL2 expression during oligodendrocyte differentiation and myelination processes.

• Investigate whether NELL2 expression in oligodendrocytes is altered in models of demyelinating diseases or in post-mortem tissue from patients with white matter disorders.

• Explore potential interactions between NELL2 and known regulators of oligodendrocyte differentiation and myelination.

The unique pattern of NELL2 puncta in oligodendrocytes (high density compared to neurons) suggests potentially distinct functions in this cell type that warrant further investigation .

How do different cell types exhibit distinct NELL2 staining patterns?

NELL2 antibody staining reveals distinct patterns across neural cell types, providing valuable insights into cell-type specific functions:

• Neural stem cells (NSCs): NELL2 appears as multiple small puncta (16-65 per cell) distributed throughout the cell body . Particularly high expression is observed in NSCs that are apically located within neural rosettes compared to cells positioned close to the basal level, as confirmed by confocal imaging of human neural rosettes double-labeled with NELL2 and ZO1 antibodies .

• Neurons: In contrast to NSCs, neurons exhibit only 1-5 NELL2 puncta per cell, but these puncta are significantly larger and elongated . Approximately 94.5% of neurons show this characteristic pattern of one-two large condensed elongated NELL2 puncta .

• Oligodendrocytes: The vast majority (97.3%) of oligodendrocytes marked by CNPase exhibit a particularly high density of NELL2 puncta . This pattern is distinct from both NSCs and neurons.

• Astrocytes: GFAP-expressing astrocytes are largely devoid of NELL2 immunoreactivity .

These differential staining patterns suggest that NELL2 may serve distinct functions in different neural cell types. The dramatic reduction in puncta number but increase in puncta size during the transition from NSCs to neurons may reflect changes in NELL2 processing, storage, or secretion mechanisms as cells differentiate and mature.

Why does NELL2 appear as punctate structures rather than diffuse staining?

The punctate appearance of NELL2 staining reflects its biological distribution within cells rather than a technical artifact. NELL2 is a secreted protein that undergoes processing through the secretory pathway before being packaged into secretory vesicles. The punctate staining pattern represents these vesicular structures containing concentrated NELL2 protein .

The characteristics of these puncta vary between cell types and developmental stages: neural stem cells typically contain 16-65 small puncta distributed throughout the cell body, whereas mature neurons display only 1-5 puncta that are significantly larger and elongated . Oligodendrocytes exhibit a particularly high density of NELL2 puncta . These differences likely reflect cell-type specific mechanisms for NELL2 processing, storage, and secretion.

To confirm that punctate staining represents genuine NELL2 localization, researchers have used C-terminal EGFP-tagged NELL2 (NELL2-EGFP) to track NELL2 movement in living cells . These studies have shown that NELL2 vesicles are rapidly transported to the peripheral region of cells, consistent with its role as a secreted protein .

What are potential cross-reactivity concerns with NELL2 antibodies?

When working with NELL2 antibodies, several cross-reactivity concerns should be addressed:

• Specificity across species: While NELL2 is highly conserved, subtle differences exist between species. In research using rat NELL2 cDNA (GenBank accession number: AY089719), species-specific antibodies have been developed . Researchers should verify whether antibodies raised against one species' NELL2 will recognize NELL2 from other species.

• Discrimination between NELL1 and NELL2: NELL1 shares significant homology with NELL2, particularly in their EGF-like domains. Ensure your antibody specifically targets NELL2-unique regions to avoid cross-reactivity.

• Recognition of different NELL2 isoforms: Some NELL2 antibodies are designed to recognize specific epitopes, such as amino acids 273-588 of rat NELL2 protein . Depending on the epitope targeted, antibodies may have differential recognition of NELL2 variants or post-translationally modified forms.

• Effects of glycosylation: As NELL2 is a glycosylated protein, some antibodies may have differential affinity for glycosylated versus non-glycosylated forms.

To address these concerns, always include appropriate validation controls, such as NELL2 knockdown samples using siRNA , overexpression systems with tagged NELL2 , and comparison of results using antibodies targeting different NELL2 epitopes.

How can I optimize NELL2 immunostaining in complex tissue samples?

For optimal NELL2 immunostaining in complex tissue samples like brain organoids, consider the following methodological approach:

• Sample preparation: For brain organoids, which mimic the cellular make-up of early developing human brain, careful sectioning is crucial. Organoids should be fixed with 4% paraformaldehyde and sectioned to visualize structures like neural rosettes that are clearly evident in brightfield images .

• Antigen retrieval: For fixed tissues, include an antigen retrieval step (e.g., citrate buffer pH 6.0, 95°C for 20 minutes) to unmask epitopes that may be obscured during fixation.

• Extended permeabilization: Use 0.25-0.5% Triton X-100 for permeabilization, potentially with longer incubation times (1-2 hours) for thicker sections to ensure antibody penetration.

• Blocking optimization: Implement thorough blocking with 5% normal goat serum and 5% skim milk to reduce background staining .

• Co-staining strategy: For comprehensive analysis, pair NELL2 antibodies with markers like PAX6 and CTIP2 to identify ventricular zone and cortical plate regions , NESTIN for neural stem cells, NEUN for neurons, GFAP for astrocytes, and CNPase for oligodendrocytes .

• Imaging considerations: Use confocal microscopy for detailed analysis of NELL2 puncta distribution. When examining neural rosettes, confocal imaging of samples double-labeled with NELL2 and ZO1 antibodies can confirm the apical localization of NELL2 .

• Quantification approaches: For comparative studies, quantify both the number of NELL2-positive cells (as a percentage of total cells or specific cell types) and the characteristics of NELL2 puncta (number, size, and distribution per cell) .

How can NELL2 antibodies be used to study NELL2-receptor interactions?

NELL2 has been identified as a ligand for receptors including Robo2 and Robo3 in some contexts . To study these interactions using antibodies, several methodological approaches can be employed:

• Expression analysis: First, analyze mRNA expression of potential receptors during developmental stages of interest. For example, studies in hippocampal primary neurons have shown that Robo1-3 mRNA levels remain relatively constant throughout developmental stages .

• Ligand binding assays: Produce C-terminally tagged NELL2 (e.g., NELL2-FLAG) from transfected cells and incubate target cells with the purified protein. After washing to remove unbound NELL2, prepare whole-cell lysates and analyze by immunoblotting to assess binding. Compare binding between control cells and cells with receptors knocked down by siRNA .

• Receptor knockdown studies: Transfect cells with siRNA to knockdown putative receptors (e.g., Robo2 and Robo3) and analyze the effects on NELL2-dependent processes like neurite and axon growth . Studies in hippocampal neurons have shown that neither average neurite length nor average axon length was affected by knocking down Robo2 or Robo3 expression, suggesting that in this context, NELL2 acts through another signaling system .

• Signaling pathway analysis: Investigate downstream signaling events following NELL2 stimulation. Research has shown that inhibitors against extracellular signal-regulated kinase (ERK) dramatically inhibit NELL2-induced progression of neuronal development and axon growth , indicating ERK pathway involvement in NELL2 signaling.

What methodological approaches can detect changes in NELL2 processing or secretion?

Several methodological approaches can be used to detect changes in NELL2 processing or secretion:

• Western blotting of cellular lysates and conditioned media: Transfect cells with NELL2 expression vectors, then incubate in serum-free medium for a defined period (e.g., 2 hours). Collect both the culture medium and cell lysates, precipitate proteins from the medium using trichloroacetic acid, and perform Western blotting using anti-NELL2 antibodies or antibodies against tags (e.g., anti-GFP for NELL2-EGFP constructs) .

• Analysis of NELL2 domains: Create constructs expressing specific NELL2 domains, such as the N-terminal 29-amino acid signal peptide (amino acids 1-29) or constructs with this region deleted (amino acids 30-816) . Compare the secretion and localization patterns of these constructs to understand the role of specific domains in NELL2 processing and secretion.

• Live-cell imaging: Use C-terminal EGFP-tagged NELL2 (NELL2-EGFP) to track the movement of NELL2 in living cells . This approach allows visualization of NELL2 vesicle transport to the cell periphery, supporting its role as a secreted protein.

• Permeabilized versus non-permeabilized immunostaining: Fix cells expressing NELL2 and either permeabilize with Triton X-100 or leave untreated before immunostaining with NELL2 antibodies . Non-permeabilized cells should not show NELL2 immunoreactivity if NELL2 is primarily intracellular or secreted rather than membrane-bound.

• Subcellular fractionation: Perform subcellular fractionation to separate membrane, cytosolic, and nuclear fractions, followed by Western blotting with NELL2 antibodies . Include controls like APP-EGFP (a known membrane protein) and cytosolic EGFP for comparison.