IHC image of ab190565 staining in formalin fixed paraffin embedded tissue section of normal human cerebellum.

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 ab190565 (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^® 647 signal is dervied directly from bound ab190565. The separate images of ab190565 and DAPI alone, combined with the merged version of both signals, shows predominant co-localisation of the Alexa Fluor^® 647 signal in the nuclei of the cerebellar granule 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.

Immunohistochemistry (Frozen sections) analysis of mouse cerebellum tissue sections labeling NeuN with Purified ab190565 at 1/1000 (2.0 µg/ml).Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. DAPI was used as a counterstain.

ab190565 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 ab190565 at 1/50 dilution(shown in red) 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^® 488 Goat anti-Mouse secondary (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Immunohistochemistry (Frozen sections) analysis of rat cerebellum tissue sections labeling NeuN with Purified ab190565 at 1/1000 (2.0 µg/ml).Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. DAPI was used as a counterstain.

Effects of delayed treatment of L655,708 on neurogenesis in the peri-infarct region. Ki67, a proliferative marker, and NeuN, a neuronal marker, were used to evaluate neurogenesis in the peri-infarct region. The nuclei were counter-stained with DAPI. Asterisk represents the significant difference from vehicle (n = 8/group; p 

Panel A shown only. Rat brain sections were first pre-treated with citrate buffer (0.01 mol/L, pH 6.0) for 5 minutes at 85 °C, followed by incubation with 5% normal goat serum for 1 hour at room temperature. Sections were then incubated with anti-Ki67 (rabbit monoclonal antibody to Ki67, 1/500; ab16667) and anti-NeuN (rabbit monoclonal antibody to NeuN, 1/500; ab190565), overnight at 4°C. Sections were then rinsed in phosphate-buffered saline 3 times, followed by incubation with rabbit secondary antibody (anti-rabbit IgG, 1/1000) for 1 hour at room temperature. Fluorescence signals were then detected under a microscope. Negative control was conducted by incubating sections with PBS instead of primary antibodies. No positive signals were shown in negative control.

ab190565 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 ab190565 at 1/50 dilution (shown in red) 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^® 488 Goat anti-Mouse secondary (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

 

IHC image of ab190565 staining in formalin fixed paraffin embedded tissue section of normal rat cerebellum.

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 ab190565 (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^® 647 signal is dervied directly from bound ab190565. The separate images of ab190565 and DAPI alone, combined with the merged version of both signals, shows predominant co-localisation of the Alexa Fluor^® 647 signal in the nuclei of the cerebellar granule 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.

IHC image of ab190565 staining in formalin fixed paraffin embedded tissue section of normal mouse cerebellum.

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 ab190565 (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^® 647 signal is dervied directly from bound ab190565. The separate images of ab190565 and DAPI alone, combined with the merged version of both signals, shows predominant co-localisation of the Alexa Fluor^® 647 signal in the nuclei of the cerebellar granule 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.