IHC image of NeuN staining in a section of formalin-fixed paraffin-embedded normal human cerebral cortex 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 20mins. The section was then incubated with ab279295, 1ug/ml, for 15 mins at room temperature. A rabbit anti-mouse IgG1, ab125913, was added for 8 mins at room temperature and detected using an HRP conjugated goat anti-rabbit compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.
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 [EPR12763] (ab279295) at 1/1000 dilution

Lane 1 : Human brain tissue lysate
Lane 2 : Mouse brain tissue lysate
Lane 3 : Rat brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Exposure times: Lane 1: 3 minutes; Lane 2, 3: 11.5 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescence staining of NeuN using ab279295 in human SHSY5Y cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 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 ab279295 at 1.0 μg/ml. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and nuclear DNA was labelled with DAPI (shown in blue). The secondary only control (bottom row) was not incubated with ab279295 but otherwise processed the same. Images were acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized rat primary neural/glia cells labelling NeuN with ab279295 at 1/1000 dilution (0.1µg)/ Right compared with a Mouse monoclonal IgG isotype control/ Left.

Goat Anti-Mouse IgG (Alexa Fluor^® 647, ab150119) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescence staining of NeuN using ab279295 in human SHSY5Y cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 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 ab279295 at 1.0 μg/ml. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and nuclear DNA was labelled with DAPI (shown in blue). The secondary only control (bottom row) was not incubated with ab279295 but otherwise processed the same. Images were acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

NeuN was immunoprecipitated from 0.35 mg mouse brain tissue lysate 10 µg with ab279295 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab279295 at 1/1000 dilution. mouse IgG for IP (HRP) (ab131368) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate  10µg.

Lane 2: ab279295 IP in mouse brain tissue lysate.

Lane 3: Mouse monoclonal IgG1 (ab18443) instead of ab279295 in mouse brain tissue lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 15 seconds.