IHC image of ab190195 staining in acetone fixed  frozen tissue section of normal rat brain.

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 ab190195 (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^® 488 signal is dervied directly from bound ab190195. The separate images of ab190195 and DAPI alone, combined with the merged version of both signals, shows predominant co-localisation of the Alexa Fluor^® 488 signal in the nuclei of the hippocampal granular 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.

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.

Overlay histogram showing U-87MG cells stained with ab190195 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. 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 (ab190195, 1/500 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor® 488 used at the same concentration and conditions as the primary antibody. Unlabelled 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 antibody gave a positive signal in U-87MG fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ab190195 staining NeuN in NGF-differentiated PC12 cells (7 days). 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.