Neuronal nuclei antigen（NeuN /RBFOX3）

Structure of the NeuN target protein

Figure 1: Structure of the NeuN target protein.

Introduction to the NeuN

Protein Function

NeuN, also known as RBFOX3, is a marker that is detected exclusively in post-mitotic neurons and is highly conserved among species. NeuN was recently eventually identified as an epitope of Rbfox3, which is a novel member of the Rbfox1 family of splicing factors.
The RBFOX family comprises RBFOX1, RBFOX2, and RBFOX3, which is RNA-binding proteins that regulates alternative splicing events.
Rbfox3-dependent Numb alternative splicing plays an important role in the progression of neuronal differentiation during vbrain development.. Mutations in NeuN are associated with human neurological disorders such as neurodevelopmental delay, cognitive impairment, autism, and epilepsy.

Protein Expression

NeuN is predominantly expressed in the central nervous system and serves as a marker for post-mitotic neurons.

Protein Localization

NeuN is primarily localized in the nucleus of neurons, with partial localization in the cytoplasm.

NeuN ICC Experimental Results, Recombinant Alexa Fluor® 568 Fluorescent Anti-NeuN Antibody [EPR12763] - Neuronal Marker (ab207282)

Figure 2: NeuN ICC Experimental Results, Recombinant Alexa Fluor® 568 Fluorescent Anti-NeuN Antibody [EPR12763] - Neuronal Marker (ab207282)

Sample Name: B35 cell line

Experimental Conditions: The cells were fixed with 4% PFA (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h.

Experimental Results: Green: NeuN; Red: Tubulin; Blue: DAPI

Isoforms & Post-translational Modifications

Human (A6NFN3): Isoforms 1-2: 34-35 kDa (predicted)
Mouse (Q8BIF2): Isoforms 1-5: 34-41 kDa (predicted)
NeuN may undergo methylation modifications.

IHC Experiment Tips

Precautions

NeuN expression varies across different tissues and cells, predominantly expressed in the central nervous system. Some samples may exhibit weak or no expression. It is crucial to select appropriate experimental samples. Brain tissue is recommended as a positive control to ensure the experimental system is functioning correctly.
It is essential to include positive and negative controls in each experiment. Positive controls ensure the effectiveness of the primary and secondary antibodies. Negative controls should include the secondary antibody only, without the primary antibody.

Positive Control

Mouse or rat brain tissue

Example Results

Figure 3: Recombinant Anti-NeuN Antibody [EPR12763] - Neuronal Marker (ab177487)

Figure 3: Recombinant Anti-NeuN Antibody [EPR12763] - Neuronal Marker (ab177487)

Sample Name: Immunohistochemical fluorescence staining of mouse brain tissue (Formalin/PFA-fixed paraffin-embedded sections).

Experimental Method: Heat-mediated antigen retrieval using Tris/EDTA buffer at pH 9. To eliminate interference from previous antibody signals, heat-mediated antigen retrieval is performed between two rounds of tyramide signal amplification to prevent cross-reactivity. Sections are stained sequentially for NeuN, SOX1, and Olig2.

Experimental Results: Green: NeuN, representing neuronal cells.
Red: SOX1, representing neural progenitor cells.
Yellow: Olig2, representing oligodendrocytes.
Blue: DAPI, staining cell nuclei.

Figure 4: Recombinant Anti-NeuN Antibody [EPR21906] - Neuronal Marker (ab236870)

Figure 4: Recombinant Anti-NeuN Antibody [EPR21906] - Neuronal Marker (ab236870)

Sample Name: Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue.

Experimental Method: Heat-mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with the IHC staining protocol. Negative control with secondary antibody only: PBS was used instead of the primary antibody.

Experimental Results: Nuclear and cytoplasmic staining in neurons of the mouse cerebrum.

Recombinant Anti-NeuN Antibody [EPR21906] - Neuronal Marker (ab236870)

Figure 5: Recombinant Anti-NeuN Antibody [EPR21906] - Neuronal Marker (ab236870)

Sample Name: Immunohistochemical analysis of frozen rat cerebellum tissue.

Experimental Method: Tissue fixed with 4% PFA, followed by permeabilization with 0.2% Triton X-100. Negative control with secondary antibody only: PBS was used instead of the primary antibody.

Experimental Results: Green: NeuN; Blue: DAPI. Cytoplasmic and nuclear staining in rat cerebellum.

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample fixation:

The optimal fixation time for samples depends on the size and type of tissue, but for most samples, it is advisable to fix with 4% PFA at room temperature for 18-24 hours.
For IHC-Fr experiments using perfusion-fixed tissue, after sampling, further fixation with fixing solution for 18-24 hours is necessary, with no additional fixation required after sectioning. For fresh tissue frozen with isopentane, post-sectioning fixation with 4°C pre-chilled PFA or methanol for 10 minutes is required.
Insufficient fixation can result in higher signal at the edges of the sample and weaker or no signal in the center.
Over-fixation can mask the epitope. While antigen retrieval can help overcome this masking, if the tissue is fixed for an extended period (eg, over a weekend), there may still be no signal even after antigen retrieval.

Blocking:

When conducting experiments using secondary antibodies conjugated with fluorescent labels, it is recommended that 1% BSA and a final concentration of 0.3 M glycine be added to the blocking solution to quench the autofluorescence induced by aldehyde groups.
If using HRP conjugate for detection, treat the sections with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity.
If performing indirect detection with secondary antibodies, you should pay attention to the host species of the primary antibody and the species of the tested sample. For example, when a primary antibody from a mouse is used to detect mouse tissue, non-specific staining can easily occur. Appropriate IgG blocking reagents can be added to prevent the secondary antibody from binding to endogenous IgG, thereby reducing non-specific staining.

Antigen retrieval:

When conducting immunohistochemistry experiments on paraffin sections, we recommend heat-induced antigen retrieval at 110°C for 15 minutes using a pressure cooker.
When performing immunohistochemistry on frozen sections, if the samples have been fixed with aldehydes for 18-24 hours, you can use a microwave for short-term retrieval or enzymes for antigen retrieval. However, optimizing the enzyme concentration and retrieval time is essential to prevent damaging the tissue morphology of the sections.