RBFOX3/NeuN Antibody - BSA Free

Novus Biologicals | Catalog # NBP1-77686

Novus Biologicals
100% Guarantee

Key Product Details

Species Reactivity

Validated:

Human, Mouse, Rat, Porcine, Bovine

Cited:

Human, Mouse, Rat

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Immunohistochemistry Free-Floating, Western Blot, Flow Cytometry, Immunocytochemistry/ Immunofluorescence

Cited:

Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Immunohistochemistry Free-Floating, Western Blot, Immunocytochemistry/ Immunofluorescence, IF/IHC

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all RBFOX3/NeuN Primary Antibodies »

Product Specifications

Immunogen

A synthetic peptide made to an internal region of the human RBFOX3/NeuN protein (within residues 20-100). [Swiss-Prot A6NFN3]

Reactivity Notes

Bovine and porcine reactivity reported from a verified customer review.

Localization

Nucleus, Cytoplasm

Marker

Neuronal Marker

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

33.8 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for RBFOX3/NeuN Antibody - BSA Free

Immunohistochemistry-Paraffin: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Immunohistochemistry-Paraffin: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Immunohistochemistry-Paraffin: RBFOX3/NeuN Antibody [NBP1-77686] - IHC analysis of formalin fixed paraffin embedded tissue section from hippocampus of mouse brain with NeuN antibody at 1:200 dilution. The antibody generated a strong nuclear with weak cytoplasmic staining in NeuN reactive cells.
Immunohistochemistry-Paraffin: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Immunohistochemistry-Paraffin: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

RBFOX3-NeuN-Antibody-Immunohistochemistry-Paraffin-NBP1-77686-img0011.jpg
Immunocytochemistry/ Immunofluorescence: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Immunocytochemistry/ Immunofluorescence: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Immunocytochemistry/Immunofluorescence: RBFOX3/NeuN Antibody [NBP1-77686] - Neuro2a cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-RBFOX3/NeuN Antibody NBP1-77686 at 1 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Western Blot: RBFOX3/NeuN AntibodyBSA Free [NBP1-77686]

Western Blot: RBFOX3/NeuN AntibodyBSA Free [NBP1-77686]

Western Blot: RBFOX3/NeuN Antibody [NBP1-77686] - Western blot image submitted by a verified customer review.
Flow Cytometry: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Flow Cytometry: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Flow Cytometry: RBFOX3/NeuN Antibody [NBP1-77686] - An intracellular stain was performed on Hek293 cells with NBP1-77686AF647 (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 2.5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.
Immunocytochemistry/ Immunofluorescence: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Immunocytochemistry/ Immunofluorescence: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Immunocytochemistry/Immunofluorescence: RBFOX3/NeuN Antibody [NBP1-77686] - NeuN antibody was tested in U2OS cells with DyLight 488 (green). Nuclei and alpha-tubulin were counterstained with DAPI (blue) and DyLight 550 (red).
Immunohistochemistry: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Immunohistochemistry: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Immunohistochemistry: RBFOX3/NeuN Antibody [NBP1-77686] - Analysis of NeuN in mouse brain using DAB with hematoxylin counterstain.
Flow Cytometry: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Flow Cytometry: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686]

Flow Cytometry: RBFOX3/NeuN Antibody [NBP1-77686] - An intracellular stain was performed on U2OS cells with RBFOX3/NeuN Antibody NBP1-77686AF647 (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.
RBFOX3/NeuN Antibody - BSA Free

RBFOX3/NeuN in U-2 OS Human Cell Line.

RBFOX3/NeuN was detected in immersion fixed U-2 OS human osteosarcoma cell line using Rabbit anti-RBFOX3/NeuN Affinity Purified Polyclonal Antibody conjugated to Alexa Fluor® 488 (Catalog # NBP1-77686AF488) (green) at 10 µg/mL overnight at 4C. Cells were counterstained with DAPI (blue). Cells were imaged using a 100X objective and digitally deconvolved.
RBFOX3/NeuN Antibody - BSA Free

RBFOX3/NeuN in U-2 OS Human Cell Line.

RBFOX3/NeuN was detected in immersion fixed U-2 OS human osteosarcoma cell line using Rabbit anti-RBFOX3/NeuN Affinity Purified Polyclonal Antibody conjugated to Alexa Fluor® 647 (Catalog # NBP1-77686AF647) (green) at 10 µg/mL overnight at 4C. Cells were counterstained with DAPI (blue). Cells were imaged using a 100X objective and digitally deconvolved.
RBFOX3/NeuN Antibody - BSA Free

Immunocytochemistry/ Immunofluorescence: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686] -

Immunocytochemistry/ Immunofluorescence: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686] - Double immunofluorescence indicates that NF-kappa B & CCL5 were co-localized with microglia, astrocytes, & neurons in the ipsilateral L4–5 spinal cord on day 7 after CCI.NF-kappa B (green) colocalizes with Iba-1, GFAP, & NeuN (red) in laminae II–III of the superficial dorsal horn (A). CCL5 (red) co-localizes with Iba-1, GFAP, & NeuN (green) in the medial superficial dorsal horn (laminae II–III) (B). NF-kappa B (green) co-localizes with CCL5 (red) in laminae II–IV of the superficial dorsal horn following CCI on day 7 in the sham & CCI groups in the ipsilateral L4–5 spinal cord (C). Two single stained images (yellow) were merged. Scale = 100 μm. Image collected & cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0115120), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
RBFOX3/NeuN Antibody - BSA Free

Immunocytochemistry/ Immunofluorescence: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686] -

Immunocytochemistry/ Immunofluorescence: RBFOX3/NeuN Antibody - BSA Free [NBP1-77686] - (A) Representative Western blots depicting phosphorylated JAK3 (pJAK3), total JAK3, & corresponding beta -actin levels in the ipsilateral (CXI) versus the contralateral (CXC) cortex at 24 h in sham & mice subjected to permanent middle cerebral artery occlusion (pMCAO). (B,C) Quantified pJAK3 & JAK3 values normalized to beta -actin in the cortex of sham & mice subjected to stroke. Both pJAK3 & JAK3 are significantly increased in the ipsilateral cortex of stroked animals (*P < 0.05 & **P < 0.01 with respect to sham ipsilateral/contralateral & stroke contralateral; sham, n = 5; stroke, n = 13). Data were analyzed using a two-way ANOVA (ipsilateral/contralateral or sham/stroke) with Bonferroni post-test. (D) Diagram showing peri-infarct brain region from which images were taken (n = 3). Scale bar is 10 µm, & magnification is 60×. (E) JAK3 colocalized with microglia/macrophages labeled with Iba-1. (F) JAK3 colocalized with neurons labeled with NeuN. (G) JAK3 colocalized with endothelial cells labeled with CD31. (H) JAK3 is not colocalized with astrocytes labeled with GFAP. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28790974), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
RBFOX3/NeuN Antibody - BSA Free

RBFOX3/NeuN in U-2 OS Human Cell Line.

RBFOX3/NeuN was detected in immersion fixed U-2 OS human osteosarcoma cell line using Rabbit anti-RBFOX3/NeuN Antigen Affinity Purified Polyclonal Antibody conjugated to FITC (Catalog # NBP1-77686F) (green) at 10 µg/mL overnight at 4C. Cells were counterstained with DAPI (blue). Cells were imaged using a 100X objective and digitally deconvolved.
RBFOX3/NeuN Antibody - BSA Free

Detection of RBFOX3/NeuN in U-2 OS Human Cell Line by Flow Cytometry.

An intracellular stain was performed on U-2 OS human osteosarcoma cell line with Rabbit anti-RBFOX3/NeuN Affinity-purified Polyclonal Antibody conjugated to Phycoerythrin (Catalog # NBP1-77686PE, blue histogram) or matched control antibody (Catalog # NBP2-24983, orange histogram) at 2.5 µg/mL for 30 minutes at RT.
RBFOX3/NeuN Antibody - BSA Free

RBFOX3(NeuN) in A431 Human Cell Line.

RBFOX3(NeuN) was detected in immersion fixed A431 human skin carcinoma cell line using Rabbit anti-RBFOX3(NeuN) Affinity Purified Polyclonal Antibody conjugated to Alexa Fluor® 488 (Catalog # NBP1-77686AF488) (green) at 5 µg/mL overnight at 4C. Cells were counterstained with DAPI (blue). Cells were imaged using a 100X objective and digitally deconvolved.

Applications for RBFOX3/NeuN Antibody - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:50 - 1:100

Immunohistochemistry

1:200

Immunohistochemistry Free-Floating

reported in scientific literature (detailed protocol in PMID 22771622)

Immunohistochemistry-Frozen

reported in scientific literature (detailed protocol in PMID 22771622)

Immunohistochemistry-Paraffin

1:200

Western Blot

reported in scientific literature (PMID 26581336)
Application Notes
In ICC, nuclear staining was observed in U2OS cells. In IHC-P, staining was observed in the nuclei of mouse brain cells. Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer (pH 6.0) is recommended. IHC-Fr / IHC-FrFl application reported at 1:100 dilution with detailed protocol in PMID 22771622.

Reviewed Applications

Read 7 reviews rated 4.4 using NBP1-77686 in the following applications:

Flow Cytometry Panel Builder

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Advanced Features

  • Spectra Viewer - Custom analysis of spectra from multiple fluorochromes
  • Spillover Popups - Visualize the spectra of individual fluorochromes
  • Antigen Density Selector - Match fluorochrome brightness with antigen density
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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.05% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: RBFOX3/NeuN

Rbfox3 (also known as Fox-3, Fox1 homolog C, Hrnbp3, and D11Bwg0517e) is one of three mammalian members of the RNA-binding protein Rbfox gene family, all of which are involved in regulating alternative RNA splicing. Originally identified as an epitope, NeuN is located within the N-terminal region (6-15 amino acids) of Rbfox3. Rbfox family members are highly conserved, having a single RNA recognition motif (RRM)-type RNA binding domain (RBD) near the center of the protein sequence. The RBD amino acid (aa) sequence is identical between members, Rbfox1 and Rbfox2, with 4 differences within the 77 aa domain of Rbfox3. The fox3 gene is comprised of 15 exons in humans, 11 exons in rat, with 3 variants in mouse containing 14 exons and another 3 variants containing 15 exons. The Rbfox3 protein is highly conserved across human, mouse, and rat with 98.9% sequence identity between mouse (isoform I) and rat and 83.9% sequence identity between mouse (isoform I) and human. The expression of Rbfox3/NeuN is restricted to the nervous system and has widely been used in stroke research. It is recognized as a marker of mature neuronal cell types in the spinal cord, cerebral cortex, hippocampus, dorsal thalamus, caudate/putamen, and cerebellum. NeuN has been shown to bind DNA and is predominantly nuclear. Differences in immunoreactivity have been reported between Rbfox3/NeuN subtypes in which the 46kDa is mainly found in the nucleus whereas the 48kDa form is primarily distributed in the cytoplasm (1,2).

Rbfox proteins participate in regulation of alternative splicing between family members as well as in autoregulation. While the breadth of brain and muscle specific targets for alternative splicing is well established for Rbfox proteins, those specific to Rbfox3/NeuN along with its alternative splicing mechanism are less understood. Rbfox3 has been shown to regulate neuronal differentiation by alternative splicing of Numb pre-mRNA and has a role in adult neurogenesis. Dysfunctional Rbfox3/NeuN has been associated with various neurological disorders such as neurodevelopmental delay, autism spectrum disorder, Benign rolandic epilepsy (BRE), and cognitive impairments (1,2).

References

1. Duan W, Zhang YP, Hou Z, Huang C, Zhu H, Zhang CQ, Yin Q. (2016) Novel Insights into NeuN: from Neuronal Marker to Splicing Regulator. Mol Neurobiol. 53(3):1637-1647. PMID: 25680637.

2. Su CH, D D, Tarn WY. (2018) Alternative Splicing in Neurogenesis and Brain Development. Front Mol Biosci. 5:12. PMID: 29484299

Long Name

RNA binding fox-1 homolog 3

Alternate Names

FOX-3, FOX3, HRNBP3, NEUN, anti-NeuN, NeuN ihc, NeuN immunohistochemistry, NeuN rabbit, NeuN staining

Entrez Gene IDs

146713 (Human); 52897 (Mouse); 511773 (Bovine)

Gene Symbol

RBFOX3

UniProt

A6NFN3 (Human)

Additional RBFOX3/NeuN Products

Product Documents for RBFOX3/NeuN Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

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Product Specific Notices for RBFOX3/NeuN Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations for RBFOX3/NeuN Antibody - BSA Free

Customer Reviews for RBFOX3/NeuN Antibody - BSA Free (7)

4.4 out of 5
7 Customer Ratings
5 Stars
57%
4 Stars
29%
3 Stars
14%
2 Stars
0%
1 Stars
0%

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Customer Images

Showing  1 - 5 of 7 reviews Showing All
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  • RBFOX3/NeuN Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: retina
    Species: Rat
    Verified Customer | Posted 07/28/2020
    good reaction. Little non-specific.
    RBFOX3/NeuN Antibody - BSA Free NBP1-77686
  • RBFOX3/NeuN Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: retina
    Species: Mouse
    Verified Customer | Posted 07/28/2020
    good reaction
    RBFOX3/NeuN Antibody - BSA Free NBP1-77686
  • RBFOX3/NeuN Antibody
    Name: Suman Chaudhary
    Application: Simple Western
    Sample Tested: Adult eye and muscle tissue (labeled E and M
    Species: Human
    Verified Customer | Posted 07/21/2020
    Human retinal lysates
    RBFOX3/NeuN Antibody - BSA Free NBP1-77686
  • RBFOX3/NeuN Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: retina
    Species: Bovine
    Verified Customer | Posted 07/21/2020
    Works excellent
    RBFOX3/NeuN Antibody - BSA Free NBP1-77686
  • RBFOX3/NeuN Antibody
    Name: Ajay Ashok
    Application: Western Blot
    Sample Tested: retina
    Species: Pig
    Verified Customer | Posted 07/21/2020
    Pig retina lysates-NeuN
    RBFOX3/NeuN Antibody - BSA Free NBP1-77686
  • Name: Anonymous
    Application: Western Blot
    Sample Tested: retina
    Species: Goat
    Verified Customer | Posted 07/21/2020
    Goat retina-NeuN
    RBFOX3/NeuN Antibody - BSA Free NBP1-77686
  • RBFOX3/NeuN Antibody
    Name: Anonymous
    Application: Immunocytochemistry
    Sample Tested: mixed primary neuronal culture
    Species: Human
    Verified Customer | Posted 06/23/2020
    RBFOX3/NeuN Antibody - BSA Free NBP1-77686

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Showing  1 - 5 of 7 reviews Showing All

Protocols

View specific protocols for RBFOX3/NeuN Antibody - BSA Free (NBP1-77686):


Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.
1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.


Immunohistochemistry-Paraffin Embedded Sections
Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes.
Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in wash buffer for 5 minutes.
3. Block each section with 100-400 ul blocking solution for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Streptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in deionized water.
12. Counterstain sections in hematoxylin.
13. Wash sections in deionized water two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.
*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.

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