Anti-NeuN antibody ValidAbTM

(HB6498)
Technical documents: SDS Datasheet

Product overview

Name Anti-NeuN antibody ValidAbTM
Alternative names Fox-3
Host Rabbit
Clonality Polyclonal
Target NeuN
Description

Antibody to NeuN - marker for mature neurones expressed in the nucleus. Part of the ValidAb™ range of highly validated, data-rich antibodies.

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Validation data

Figure 1. NeuN and parvalbumin expression in rat CA1 visualised using HB6498

HB6498 stains the NeuN expressing neurones of rat CA1 while HB6457 (mouse monoclonal anti-parvalbumin) stains the subset expressing parvalbumin. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:5000 dilution, 0.2µg/ml) and HB6457 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with polyclonal goat anti-mouse DyLight 488 conjugated (Thermofisher 35503, 1:300 dilution) and polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) secondary antibodies. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured as a tilescan using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 20x objective and A4 (100ms exposure @ 1.7x gain) / GFP (161ms exposure @3x gain) / TX2 (410ms @ 4.6x gain) filters. Images were captured as a stack (3.4µm z-spacing) before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 2. NeuN expressing neurons in the rat hippocampus visualised using HB6498

HB6498 stains the NeuN expressing neurones found within the rat hippocampal formation. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:2000 dilution, 0.5µg/ml). This was followed by a two hour incubation with a polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) secondary antibody. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured as a tilescan using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI (26ms exposure @ 5.8x gain) / Y3 (177ms exposure @ 5.4x gain) filters. Images were captured as a stack (5.4µm z-spacing) before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 3. NeuN expressing neurones in the rat dentate gyrus visualised with HB6498 conjugated to Janelia Fluor 525.

HB6498 conjugated to Janelia Fluor 525 (HB8455) stains the NeuN expressing neurons of the dentate gyrus. Method: HB6498 was conjugated to Janelia Fluor 525 SE (for protocol see HB8455). Rat brains were then dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (4µg/ml) DAPI (HB0747) was then incubated at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI (17ms exposure) / TX2 (200ms exposure) filters. Images were captured as a stack (4.3µm z-spacing) before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 4. Concentration response of HB6498 staining in rat hippocampal sections.

HB6498 stains NeuN expression neurones in the hippocampus at concentrations as low as 62.5ng/ml (1 in 16,000 dilution). Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in dilutions of HB6498 ranging from 1 in2000 (500ng/ml) to 1 in 16,000 (62.5ng/ml). This was followed by a two hour incubation with a polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) secondary antibody. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI/TX2 filters. Exposure times were:

  • 1 in 2,000 dilution: DAPI: 314ms, TX2: 113ms
  • 1 in 4,000 dilution: DAPI: 314ms, TX2: 240ms
  • 1 in 8,000 dilution: DAPI: 314ms, TX2: 240ms
  • 1 in 16,000 dilution: DAPI: 314ms, TX2: 240ms


Images were captured as a stack (6.1µm z-spacing) before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 5. NeuN expression in various tissue lysates and preparations.

HB6498 revealed the 76kDa (Synapsin I), 50kDa (FOX3) and 45kDa (FOX3) bands associated with the NeuN antigen. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour, 1610374) before being run at 60V for 30 minutes followed by 100V for 80 minutes then 130V for 40 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6498 at a 1:1,000 dilution (1µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) and HB8224 (rabbit polyclonal anti-ß-actin, 1:1000 dilution) overnight at 4°C. Following washing the membrane was incubated in secondary antibodies (1:10,000 dilution for both, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682 and Polyclonal Goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure).

Figure 6. Concentration response of HB6498 staining in a rat brain cytosol preparation.

HB6498 shows strong affinity for NeuN with bands visible at as low as 0.031µg/ml (1 in 32,000 dilution). At very high concentrations an additional band at ≈27kDa is visible which is not stained at dilutions below 1in2000. Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples were loaded (20µg / lane) onto a 12% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour, 1610374) before being run at 60V for 30 minutes followed by 120V for 100 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6498. Each strip was incubated separately with a separate HB6498 concentration with this ranging from 4µg/ml (1:250 dilution) to 0.031µg/ml (1:32,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.

Figure 7. Independent antibody validation of HB6498 in rat cortex.

HB6429 (mouse monoclonal anti-NeuN) and HB6498 (rabbit polyclonal anti-NeuN) show overlapping patterns of staining in rat cortex providing strong evidence for specificity. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:5000 dilution, 0.2µg/ml) and HB6429 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with polyclonal goat anti-mouse DyLight 488 conjugated (Thermofisher 35503, 1:300 dilution) and polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) secondary antibodies. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 20x objective and DAP (10ms exposure) / L5 (435ms exposure) / TX2 (118ms exposure) filters. Images were captured as a stack before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 8. NeuN expressing neurons visualised with HB6498 conjugated to Janelia Fluor 525.

HB6498 conjugated to Janelia Fluor 525 (HB8455) stains NeuN expressing neurons in the rat brain. Method: HB6498 was conjugated to Janelia Fluor 525 SE (for protocol see HB8455). Rat brains were then dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (4µg/ml) DAPI (HB0747) was then incubated at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 20x objective and DAPI (8ms exposure) / TX2 (150ms exposure) filters. Images were captured as a stack (0.6µm z-spacing) before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 9. NeuN expressing neurons in the rat dentate gyrus visualised using HB6498

HB6498 stains the NeuN expressing neurones found within the rat hippocampal formation. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% goat serum before incubation overnight in HB6498 (1:2000 dilution, 0.5µg/ml). This was followed by a two hour incubation with a polyclonal goat anti-rabbit DyLight 594 (Thermofisher 35561) secondary antibody. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. The image was captured using a Leica DMI6000B inverted epifluorescence microscope coupled to a Photometric Prime 95B camera utilising a 10x objective and DAPI (314ms exposure) / TX2 (113ms exposure) filters. Images were captured as a stack (4.3µm z-spacing) before being deconvolved using Huygens Professional software then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Product information

Immunogen

Amino acids 5 - 24 of human FOX3 expressed and purified from E. coli

Epitope Amino acids 5-24 of human NeuN
Isotype IgG
Purification

Immunogen affinity purification

Concentration 1mg/ml
Formulation 50% PBS, 50% glycerol + 5mM sodium azide
Predicted species reactivity Mouse, Rat, Human
Tested species reactivity Mouse, Rat

Tested applications

Applications WB, IHC(IF)
Western blot optimal concentration

0.5µg/ml (1:2000 dilution) as tested in a rat brain cytosol preparation

IHC(IF) optimal concentration

0.5µg/ml (1:2000 dilution) as tested in rat brain sections

Positive control

NeuN is highly expressed in the neurons of the CNS and PNS. It is also expressed in SH-SY5Y cells.

Negative control

Any tissue not of neural origin. Most cell lines are NeuN negative.

Open data link

Please follow this link to OSF

Target information

Other names

FOX3, RNA binding protein fox-1 homolog 3, Fox-1 homolog C, RBFOX3, RFOX3

UniProt ID A6NFN3
Structure image  Chemical Structure
Gene name RBFOX3
NCBI full gene name RNA binding fox-1 homolog 3
Entrez gene ID

146713

Amino acids

Dependent on isoform

Isoforms

NeuN binds primarily to FOX3 which has two isoforms. Isoform 1 is described as the canonical sequence with 312 amino acids (33.8kDa) while isoform 2 has a 13 residue insert at position 312 leading to a total length of 325 amino acids (35.1kDa). NeuN antibodies also bind to synapsin-1 in western blot experiments (but not in IHC or ICC) which has two isoforms. Isoform 1 is 705aa long (74.1kDa) while isoform 2 is shorter at 669aa (70.0kDa).

Expression

NeuN is expressed only within neurones. While the vast majority of neurones express NeuN some cell types such as Purkinje cells, stellate and golgi cells do not show immunoreactivity.

Subcellular expression

Expression is primarily localised to the nucleus however some FOX3 isoforms can localise to the cytosol.

Processing

None

Post translational modifications

Phosphorylation has been reported (see Lind et al., 2004. J Neurosci Res. 79: 295-302) which is directly related to immunoreactivity whereby dephosphorylation abolished staining.

Homology (compared to human)

Mouse FOX3 shows 95.02% identity to human FOX3 wheras rat FOX3 shows no similarity due to a large 47 residue insertion at amino acid 252 in rats.

Similar proteins

RNA-binding protein fox-1 homolog 1 (40-44kDa) shows 67.3% identity while RNA-binding protein fox-1 homolog 2 (37-47kDa) shows 56.5% identity

Storage & Handling

Storage instructions

-20°C

Important This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use

FAQs

What mounting media do you recommend to use with this antibody?
What other neuroscience markers are available?
Are all neurons NeuN positive?

While the vast majority of neurones express NeuN some subtypes such as Purkinje cells, stellate and golgi cells do not show immunoreactivity. NeuN is also not expressed in immature neurones.

What guarantee do you have that my NeuN antibody will perform as expected?

We guarantee that your NeuN antibody will work for the applications and species we list on the datasheet. If the antibody fails to perform as expected then we are happy to offer a 100% refund guarantee. For more details please see our guarantee policy.

Will my NeuN antibody work against species that have not been listed on the datasheet?

A species not being listed doesn’t mean that the antibody won’t work, just that we haven’t tested it. If you test one of our antibodies in a new species please let us know (positive or negative)!

What protocols are available for use with this NeuN antibody

We have made a comprehensive collection of protocols that we have used in our experiments to validate this NeuN antibody.

What counterstains do you recommend for use in ICC and IHC with this NeuN antibody?

We recommend using either DAPI or Hoechst 33342 to label cell nuclei. In some experiments it is also helpful to label actin filaments in the cytoskeleton using a Phalloidin conjugate such as FITC Phalloidin or Rhodamine Phalloidin-TRITC.

Any other questions?

For any other questions about our antibody products please see our technical FAQs for antibodies

References for Anti-NeuN antibody ValidAbTM

References are publications that support the biological activity of the product
  • Novel Insights into NeuN: from Neuronal Marker to Splicing Regulator.

    Duan W et al (2016) Molecular neurobiology 53 : 1637-1647
  • NeuN As a Neuronal Nuclear Antigen and Neuron Differentiation Marker.

    Gusel'nikova VV et al (2015) Acta naturae 7 : 42-7
  • Identification of neuronal nuclei (NeuN) as Fox-3, a new member of the Fox-1 gene family of splicing factors.

    Kim KK et al (2009) The Journal of biological chemistry 284 : 31052-61
  • Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization.

    Lind D et al (2005) Journal of neuroscience research 79 : 295-302
  • NeuN: a useful neuronal marker for diagnostic histopathology.

    Wolf HK et al (1996) The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 44 : 1167-71