Immunofluorescence staining of NeuN using ab279297 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 ab279297 at 1.0 μg/ml. Cells were then incubated with ab150165, Goat Anti-Rat 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 ab279297 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.

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 ab279297, 1ug/ml, for 15 mins at room temperature. A rabbit anti-rat IgG2a, ab102248, 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.

Immunofluorescence staining of NeuN using ab279297 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 ab279297 at 1.0 μg/ml. Cells were then incubated with ab150165, Goat Anti-Rat 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 ab279297 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.

All lanes : Anti-NeuN antibody [EPR12763] (ab279297) 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 : Goat Anti-Rat IgG H&L (HRP) (ab205720) at 1/5000 dilution

Exposure time: Lane 1: 48 seconds; Lane 2, 3: 4.5 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized rat primary neural/glia cells labelling NeuN with ab279297 at 1/1000 dilution (0.1µg)/ Right compared with a Mouse monoclonal IgG isotype control/ Left.

Goat F(ab)2 Anti-Rat IgG Fc (Alexa Fluor^® 647, ab150163) at 1/2000 dilution was used as the secondary antibody.