Alexa Fluor® 647 Anti-Synaptophysin antibody [YE269] - Synaptic Marker

• Flow Cyt

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Flow Cytometry - Alexa Fluor® 647 Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB196166)

Overlay histogram showing PC12 cells stained with ab196166 (red line). The cells were fixed with 4% formaldehyde (10 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 (ab196166, 1/500 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor® 647 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 solid-state 25mW red diode laser (635 nm) and 675/30 bandpass filter.

This antibody gave a positive signal in PC12 fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 647 Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB196166)

ab196166 staining Synaptophysin in NGF-differentiated PC-12 cells. The cells were fixed with 100% methanol (5 min), permeabilised in 0.1% Triton X-100 for 5 minutes 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 ab196166 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 1/167 dilution overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

This product gave a positive signal in 5% formaldehyde (10 min) fixed NGF-differentiated PC-12 cells under the same testing conditions.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Alexa Fluor® 647 Anti-Synaptophysin antibody [YE269] - Synaptic Marker (AB196166)

Image A : composite multiplex immunohistochemistry staining using conjugates panel composed of anti-NeuN (Alexa Fluor^® 488) (ab190195, 1/100 dilution, 5 µg/mL) (green), anti-GFAP (Alexa Fluor^® 555) (ab201735, 1/100 dilution, 5 µg/mL) (magenta) and anti-Synaptophysin (Alexa Fluor^® 647) (ab196166, 1/100 dilution, 5 µg/mL) (white) on Formalin/PFA-fixed paraffin-embedded sections of rat cerebellum. Nuclear DNA was labelled with DAPI (blue).

Image B : Formalin/PFA-fixed paraffin-embedded section of rat cerebellum stained with anti-NeuN (Alexa Fluor^® 488) (ab190195, 1/100 dilution, 5 µg/mL).

Image C : Formalin/PFA-fixed paraffin-embedded section of rat cerebellum stained with anti-GFAP (Alexa Fluor^® 555) (ab201735, 1/100 dilution, 5 µg/mL).

Image D : Formalin/PFA-fixed paraffin-embedded section of rat cerebellum stained with anti-Synaptophysin (Alexa Fluor^® 647) (ab196166, 1/100 dilution, 5 µg/mL).

Immunohistochemistry staining was performed on a Leica Biosystems BOND^® RX instrument. Heat mediated antigen retrieval with EDTA (pH 9.0, 110°C) for 40 mins. The section was then incubated at room temperature for 1 hour and mounted using Dako Fluorescence Mounting Medium^®.

Image acquisition was performed with Leica SP8 confocal microscope. For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.