Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis ofMouse cerebrumtissue labelling NeuN with ab177487 at 1/100 dilution (B), SOX1 with ab242125at 1/100 dilution (C) and Olig2 with ab109186at 1/100 dilution (D).Anti-Rabbit and Mouse Polymer HRP was used as a secondary antibody, and DAPI was used for a nuclear counter stain.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins. Heat mediated antigen retrieval (Leica ER2, PH9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibodies from the previous round, to avoid any cross-reactivity.

Panel A: merged staining of anti- NeuN (green, Opal™520), anti-SOX1 (red, Opal™570) and anti- Olig2 (yellow, Opal™690).

Panel B: anti-NeuN stands for neurons.

Panel C: anti-SOX1 stained on neural progenitors.

Panel D: anti-Olig2 stained on oligodendrocyte.

The section was incubated in three rounds of staining: in the order of ab177487, ab242125 and ab109186 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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.

Immunofluorescence staining of NeuN using ab177487 in ioNEURONS/glut cells (Human iPSC-Derived Glutamatergic Neurons, ab259259), which were differentiated for 1 day post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab177487 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

Immunocytochemistry/immunofluorescence analysis of Mouse primary neuron cells labelling NeuN with ab177487 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/1000 was used as the secondary antibody (green). Cells were counterstained with Anti-MAP2 mouse monoclonal antibody (ab11267) at 1/200 dilution and visualised using Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) at 1/1000 dilution (red). Nuclear DNA was labelled with DAPI (blue).

Confocal image showing mainly nuclear staining in mouse primary neuron cells. Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection.

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 ab150097Goat anti-rabbit IgGconjugated to Alexa Fluor^® 488 (1/500) and ab150176Goat anti-chicken IgY conjugated to Alexa Fluor^® 594 (1/500)

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 protocolsdesigned 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.

Immunocytochemistry/immunofluorescence analysis ofhuman neurons differentiated from iPSCs labelling NeuN (green) with ab177487 at 1/500 in 0.1% TritonX-100, 1% goat serum, 1X PBS for 16 hours at 4°C. Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100. Then, cells were blocked with 5% serum for 20 minutesat 23°C. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/1000 was used as the secondary antibody. Tuj1 antibody was used to stain neuronal dendrites and axons (red).

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

Competitor A: Leading mouse monoclonal.

Competitor B: Non-Abcamrabbit 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.

IHC image ofNeuN (ab177487) with Anti-Rabbit IgG VHH Single Domain Antibody (HRP) (ab191866) staining in formalin fixed paraffin embedded normal humancerebellum 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.

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 platedout 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 μlab177487 (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 withab177487 (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).

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/1x10⁶cells 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.

Alexa Fluor^® 488 (ab190195) and Alexa Fluor^® 647 (ab190565) conjugated versions are available for this clone.

Immunocytochemsitry/Immunofluorescence analysis of SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling NeuN (green) with 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).

Exposure time -Lane 1-2: 3 minutes.Lane 3-4: 1 minute.

Blocking and dilution buffer: 5% NFDM/TBST.

​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.

ab177487 stainingNeuN in mousefree floating 50 micron lumbar spinal cord tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with formaldehyde, permeabilized withTriton X-100 and blocked with 10%serum for2 hoursat 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.

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IHC-Fr staining of NeuN on zebrafish braintissue 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°C. ab177487 was diluted 1/100 and incubated for 16 hours at 4°C. The secondary antibody used was anti rabbit IgG conjugated to Alexa Fluor^® 488 (1/1000). DAPI used as counterstain.

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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 inblocking 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.

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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°C. ab177487 was diluted 1/1000 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).

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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°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).

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gliocytoma tissue labelling NeuN with 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.

IHC-P image of FOX3/NeuN staining onrat 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).

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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 diluted1/800 and incubated with the sectionsfor 2 hours at 21°C. The secondary antibody used was goat polyclonal to rabbit IgG conjugated to biotin (1/250).

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IHC-P image of FOX3/NeuN staining onzebrafish spinal cordsections 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).

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IHC-P image of FOX3/NeuN staining onmarmoset cerebellumsections 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).

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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°C. ab177487 was diluted 1/1000 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).

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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°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).

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