ab104224 staining NeuN - Neuronal Marker in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes 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 ab104224 at 0.1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

IHC image of NeuN staining in rat brain formalin-fixed paraffin-embedded tissue section, performed on a Leica Bond™ system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab104224, 1 µg/ml, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

 

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

 

All lanes : Anti-NeuN antibody [1B7] - Neuronal Marker (ab104224) at 1/1000 dilution

Lane 2 : Adult rat whole brain lysate
Lane 3 : Embryonic E20 rat whole brain lysate
Lane 4 : Adult mouse whole brain lysate

Predicted band size: 46, 48 kDa

Rat brain neural cultures stained with ab104224 in pink, with ab4674 (chicken polyclonal to GFAP)  in green and DNA in blue. ab104224 reveals strong nuclear and distal cytoplasmic staining. It does not stain astrocytes and other non-neuronal cells.

IHC image of NeuN staining in mouse cerebellum formalin-fixed paraffin-embedded tissue section, performed on a Leica Bond™ system using the standard protocol B.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab104224, 1 µg/ml, for 15 minutes at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

 

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

Immunofluorescent analysis of rat brain stem co-stained with ab104224 in green, and a chicken pAb to microtubule associated protein 2 (MAP2) in red. Blue is DAPI staining of nuclear DNA.

Following transcardial perfusion with 4% paraformaldehyde, the brain was post fixed for 24 hours, cut to 45μM, and free-floating sections were stained. The Fox3/NeuN antibody selectively stains nuclei and the proximal cytoplasm of neuronal cells while the MAP2 antibody labels dendrites and overlaps with Fox3/NeuN staining in the perikarya of neurons.

IHC image of FOX3/NeuN staining in human normal hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab104224, 5 µg/ml, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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