Figure 1. NeuN expressing neurons in the dentate gyrus visualised using HB9493.
HB9493 staining reveals the dense neuronal layers in the rat dentate gyrus. 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 transverse 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 HB9493 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with a polyclonal donkey anti-goat DyLight 594 conjugated secondary antibody (Thermofisher SA5-10088, 1:300 dilution). 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 (10ms exposure) / TX2 (89ms 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 2. Concentration response of HB9493 staining in rat dentate gyrus.
HB9493 labels neurons found in the dentate gyrus at concentrations as low as 125ng/ml (1:8,000 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 transverse 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 differing concentrations of HB9493 ranging from 1µg/ml (1:1,000 dilution) to 0.125µg/ml (1:8,000). This was followed by a two hour incubation with a polyclonal donkey anti-goat DyLight 594 conjugated secondary antibody (Thermofisher SA5-10088, 1:300 dilution). 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:
1µg/ml – DAPI: 10ms, TX2: 89ms
0.5µg/ml – DAPI: 10ms, TX2: 59ms
0.25µg/ml – DAPI: 10ms, TX2: 135ms
0.125µg/ml – DAPI: 10ms, TX2: 107ms
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 3. Independent antibody validation of HB9493 in rat hippocampus.
The staining patterns of HB6429 (mouse monoclonal anti-NeuN) and HB9493 (goat polyclonal anti-NeuN) overlap showing 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 transverse 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 HB9493 (1:1000 dilution, 1µg/ml) and HB6429 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with polyclonal donkey anti-goat DyLight 594 conjugated (Thermofisher SA5-10088, 1:300 dilution) and donkey anti-mouse DyLight 488 conjugated (Thermofisher SA5-10166, 1:300 dilution) 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 DAPI (10ms exposure) / L5 (249ms exposure) / TX2 (118ms exposure) filters. Images were captured as a stack (0.98µm 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 1. NeuN expressing neurons in the dentate gyrus visualised using HB9493.
HB9493 staining reveals the dense neuronal layers in the rat dentate gyrus. 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 transverse 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 HB9493 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with a polyclonal donkey anti-goat DyLight 594 conjugated secondary antibody (Thermofisher SA5-10088, 1:300 dilution). 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 (10ms exposure) / TX2 (89ms 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 2. Concentration response of HB9493 staining in rat dentate gyrus.
HB9493 labels neurons found in the dentate gyrus at concentrations as low as 125ng/ml (1:8,000 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 transverse 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 differing concentrations of HB9493 ranging from 1µg/ml (1:1,000 dilution) to 0.125µg/ml (1:8,000). This was followed by a two hour incubation with a polyclonal donkey anti-goat DyLight 594 conjugated secondary antibody (Thermofisher SA5-10088, 1:300 dilution). 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:
1µg/ml – DAPI: 10ms, TX2: 89ms
0.5µg/ml – DAPI: 10ms, TX2: 59ms
0.25µg/ml – DAPI: 10ms, TX2: 135ms
0.125µg/ml – DAPI: 10ms, TX2: 107ms
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 3. Independent antibody validation of HB9493 in rat hippocampus.
The staining patterns of HB6429 (mouse monoclonal anti-NeuN) and HB9493 (goat polyclonal anti-NeuN) overlap showing 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 transverse 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 HB9493 (1:1000 dilution, 1µg/ml) and HB6429 (1:1000 dilution, 1µg/ml). This was followed by a two hour incubation with polyclonal donkey anti-goat DyLight 594 conjugated (Thermofisher SA5-10088, 1:300 dilution) and donkey anti-mouse DyLight 488 conjugated (Thermofisher SA5-10166, 1:300 dilution) 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 DAPI (10ms exposure) / L5 (249ms exposure) / TX2 (118ms exposure) filters. Images were captured as a stack (0.98µm 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
N-terminal 100 amino acids of human FOX3 expressed and purified from E. coli
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
IHC(IF)
IHC(IF) optimal concentration
1:1000 (1μg/ml) as assessed 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.
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
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.
Western Blot Protocol (1 MB) 
