Alexa Fluor® 647 Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker (AB225381)

Overlay histogram showing A673 cells stained with ab225381 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab225381, 1/500000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Alexa Fluor^® 647 (ab199093) 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 40 mW Red laser (640nm) and 670/14 bandpass filter.

This antibody gave a positive signal in A673 cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

• ICC

Unknown

Immunocytochemistry - Alexa Fluor® 647 Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker (AB225381)

ab225381 staining Caveolin-1 in wild-type HeLa cells (top panel) and CAV1 knockout HeLa cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes 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 with ab225381 at 1/500 dilution and ab195887 (Mouse monoclonal to alpha Tubulin - Alexa Fluor^® 488) at 1/250 dilution overnight at 4°C. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker (AB225381)

Flow cytometry overlay histogram showing wild-type (green line) and CAV1 knockout (ab255371) HeLa cells stained with ab225381 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab225381) (1x 10⁶ in 100μl at 0.008μg/ml (1/62500) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Alexa Fluor^® 647 (ab199093) used at the same concentration and conditions as the primary antibody (wild-type - black line knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 40 mW Red laser (640nm) and 660/10 bandpass filter.

• ICC

Lab

Immunocytochemistry - Alexa Fluor® 647 Anti-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker (AB225381)

ab225381 staining Caveolin-1 in A673 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes 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 ab225381 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

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

This product also gave a positive signal under the same testing conditions in A673 cells fixed with 4% formaldehyde (10 min).