Clone EPR12763 (ab209898) has been successfully conjugated by Abcam. This image was generated using Anti-NeuN antibody [EPR12763] - Neuronal Marker (Alexa Fluor® 488). Please refer to ab190195 for protocol details.

ab190195 staining NeuN in U87-MG cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab190195 at 1/50 dilution (shown in green) and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin) at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an Alexa Fluor^® 594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.

This data was developed using the same antibody clone in a different buffer formulation (ab177487).

Fluorescence multiplex immunohistochemical analysis of human cerebellum tissue (formalin-fixed paraffin-embedded section).

Merged staining of Neu-N (ab177487; yellow; Opal™570), anti-beta III Tubulin (ab52623; red; Opal™690) and anti-GFAP (ab68428; green; Opal™520).

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ kit.

The section was incubated in three rounds of staining with ab177487 (1/1000 dilution), ab52623 (1/200 dilution) and ab68428 (1/250 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (pH 6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

DAPI (blue) was used as a nuclear counter stain.

IHC image of NeuN (ab177487) with Anti-Rabbit IgG VHH Single Domain Antibody (HRP) (ab191866) staining in formalin fixed paraffin embedded normal human cerebellum tissue section.

The section was dewaxed and then pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked using in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1 hour at room temperature. The section was then incubated with rabbit monoclonal antibody [EPR12763] to NeuN (ab177487, 0.1µg/ml) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. Endogenous peroxidases were quenched using 1.6% (v/v) hydrogen peroxide in TBS containing 0.025% (v/v) Triton X-100 for 30 minutes at room temperature, with agitation. The secondary antibody, Anti-Rabbit IgG VHH Single Domain Antibody (HRP) (ab191866, 1.0µg/ml) was then applied for 1 hour at room temperature in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA before being developed for 10 minutes at room temperature using Steady DAB/Plus (ab103723). The section was then counterstained with hematoxylin and mounted with DPX.

The negative control (secondary antibody only, no primary) inset shows no staining, demonstrating secondary antibody specificity.

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

Immunocytochemsitry analysis of neurons labeling nuclei with NeuN.

Primary cortical neurons were prepared from the cortices of 1-day-old newborn pups. Briefly, the cortices were dissected in cold PBS. Tissues were collected and washed in PBS, and 0.05% (v/v) trypsin was added for digestion at 37°C for 15 minutes. The digestion was stopped by the addition of fetal bovine serum to a final concentration of 10% (v/v). Cells were collected by centrifugation at 800 x g for 10 minutes to remove the PBS and were resuspended in Neurobasal medium supplemented with 2% (v/v) B27.

Neurons plated out were rinsed with PBS three times and then fixed in 4% paraformaldehyde for 25 minutes at 4°C. Fixed cells were incubated in 0.1% (v/v) sodium citrate contain 0.1% (v/v) Triton X-100 for 2 minutes on ice, and then neurons were washed twice with PBS and incubated with 300 μl ab177487 (1:500 in PBS containing 10% goat serum) for 2 hours at 37°C. An Alexa-Fluor^® 594-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (4',6-diamidino-2-phenylindole) was added for 10 minutes at room temperature followed by PBS washing to fluorescently label nuclei. Samples were photographed using a fluorescence microscope (LEICA DMI3000, Japan) and analysed using the Leica application suite.

Neuronal nuclei were labeled with ab177487 (top left panel, red), while all cells nuclei were stained in blue (top right panel).

(Purity of cultured mouse cortical neuron was 93.00% ± 1.23% at 7 days in vitro).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

Overlay histogram showing U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells stained with ab177487 (red line).

The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab177487, 1/100 dilution) for 30 minutes at 22ºC. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 1μg/1x10⁶ cells used under the same conditions. Unlabeled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

Immunocytochemsitry/Immunofluorescence analysis of SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling NeuN (green) with purified ab177487 at 1/300. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

Immunocytochemsitry/Immunofluorescence analysis of SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labelling NeuN (green) with unpurified ab177487 at 1/80. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

Clone EPR12763 (ab209898) has been successfully conjugated by Abcam. This image was generated using Anti-NeuN antibody [EPR12763] - Neuronal Marker (Alexa Fluor® 647). Please refer to ab190565 for protocol details.

IHC image of ab190565 staining in formalin fixed paraffin embedded tissue section of normal human cerebellum.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked using in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1h at room temperature. The section was then incubated with ab190565 (1/50) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. The section was then counterstained and mounted with SlowFade^® Gold Antifade Mountant with DAPI.

The DAPI only control (no antibody) inset shows no autofluorescence, demonstrating that any Alexa Fluor^® 647 signal is dervied directly from bound ab190565. The separate images of ab190565 and DAPI alone, combined with the merged version of both signals, shows predominant co-localisation of the Alexa Fluor^® 647 signal in the nuclei of the cerebellar granule layer.

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gliocytoma tissue labelling NeuN with purified ab177487 at 1/3000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gliocytoma tissue labelling NeuN with unpurified ab177487 at 1/800. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

?An independent comparison of commercially available NeuN clones in IHC-Fr (acetone-fixed mouse dentate gyrus sections).

Competitor A: Leading mouse monoclonal.

Competitor B: Non-Abcam rabbit monoclonal.

ab177487 produces intense, specific staining with minimal background, even at half the dilution of competing antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-Fr staining of NeuN on zebrafish brain tissue at 4 days post-fertilization using ab177487 (1/100). The sections were fixed in paraformaldehyde and permeabilized using triton X. Antigen retrieval uisng sodium citrate was used. The sections were blocked using 5% BSA for 1 hour at 23^oC. ab177487 was diluted 1/100 and incubated for 16 hours at 4^oC. The secondary antibody used was anti rabbit IgG conjugated to Alexa Fluor^© 488 (1/1000). DAPI used as counterstain.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

An independent comparison of commercially available NeuN clones in IHC-P.

Competitor A: Leading mouse monoclonal.

Competitor B: Non-Abcam rabbit monoclonal.

Sodium citrate was used for antigen retrieval in all 3 samples.

ab177487 produces specific staining, equivalent to the leading mouse monoclonal at half the dilution. The non-Abcam mouse monoclonal was less specific as it stained Purkinje cells, which do not express NeuN.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

ab177487 staining NeuN in mouse brain tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with formaldehyde and blocked with Triton X-100 + 0.4% horse seurm for 30 minutes at 20°C. Samples were incubated with primary antibody (1/500 in blocking solution) for 16 hours at 4°C. An Alexa Fluor^® 594-conjugated donkey anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-P image of NeuN (green) and GFAP (red) double staining on mouse cerebellum sections using ab177487 (1/5000) and ab4674 (1/1500) respectively.
The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were then incubated with Rabbit Monoclonal to NeuN (ab177487) diluted at 1/5000 and Chicken Polyclonal to GFAP (ab4674) diluted at 1/1500. The primary antibody was detected using ab150097 Goat anti-rabbit IgG conjugated to Alexa Fluor^© 488 (1/500) and ab150176 Goat anti-chicken IgY conjugated to Alexa Fluor^© 594 (1/500)
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

This data was developed using ab177487, the same antibody clone in a different buffer formulation.

NeuN antibody ab177487 was used with Tissue Clearing Kit ab243298 to penetrate, stain and clear a 1 mm coronal section of mouse brain. Blue: DAPI, Green: NeuN.

Learn more about tissue clearing kits, reagents, and protocols designed to make it easier to stain thick tissue sections and get more data from each valuable tissue section.

For 1 mm brain sections, we recommend a starting dilution of 1:200, and also using Goat Anti-Rabbit IgG H&L AlexaFluor488 (ab150077) at a dilution of 1:400.

ab177487 staining NeuN in mouse free floating 50 micron lumbar spinal cord tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with formaldehyde, permeabilized with Triton X-100 and blocked with 10% serum for 2 hours at 25°C. Samples were incubated with primary antibody (1/500 in PBS + Triton) for 16 hours at 4°C. An Alexa Fluor^® 594-conjugated donkey anti-rabbit IgG polyclonal (1/700) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-P image of FOX3/NeuN staining on cat cerebellum sections using ab177487 (1/1000).
Sections were de-paraffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 minutes at 21^oC. ab177487 was diluted 1/1000 and incubated with the sections for 2 hours at 21^oC. The secondary antibody used was goat polyclonal to rabbit IgG conjugated to biotin (1/250).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-P image of FOX3/NeuN staining on dog cerebellum sections using ab177487 (1/500).
Sections were de-paraffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 minutes at 21^oC. ab177487 was diluted 1/500 and incubated with the sections for 2 hours at 21^oC. The secondary antibody used was goat polyclonal to rabbit IgG conjugated to biotin (1/250).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-P image of FOX3/NeuN staining on sheep brain (Frontal cortex) sections using ab177487 (1/1000). Sections were de-paraffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 minutes at 21^oC. ab177487 was diluted 1/1000 and incubated with the sections for 2 hours at 21^oC. The secondary antibody used was goat polyclonal to rabbit IgG conjugated to biotin (1/250).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-P image of FOX3/NeuN staining on goat cerebellum sections using ab177487 (1/500). Sections were de-paraffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 minutes at 21^oC. ab177487 was diluted 1/500 and incubated with the sections for 2 hours at 21^oC. The secondary antibody used was goat polyclonal to rabbit IgG conjugated to biotin (1/250).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-P image of FOX3/NeuN staining on marmoset cerebellum sections using ab177487 (1/2000). Sections were de-paraffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 minutes at 21°C. ab177487 was diluted 1/2000 and incubated with the sections for 2 hours at 21°C. The secondary antibody used was goat polyclonal to rabbit IgG conjugated to biotin (1/250).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-P image of FOX3/NeuN staining on zebrafish spinal cord sections using ab177487 (1/500). Sections were de-paraffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 minutes at 21°C. ab177487 was diluted 1/500 and incubated with the sections for 2 hours at 21°C. The secondary antibody used was goat polyclonal to rabbit IgG conjugated to biotin (1/250).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-P image of FOX3/NeuN staining on rat brain (SVZ) sections using ab177487 (1/2000). Sections were de-paraffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 minutes at 21°C. ab177487 was diluted 1/2000 and incubated with the sections for 2 hours at 21°C. The secondary antibody used was goat polyclonal to rabbit IgG conjugated to biotin (1/250).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).

IHC-P image of FOX3/NeuN staining on mouse brain (frontal cortex) sections using ab177487 (1/800). Sections were de-paraffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 minutes at 21°C. ab177487 was diluted 1/800 and incubated with the sections for 2 hours at 21°C. The secondary antibody used was goat polyclonal to rabbit IgG conjugated to biotin (1/250).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177487).