Flow Cytometry (Intracellular) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)

Overlay histogram showing SV40LT-SMC cells stained with ab7817 (red line). The cells were fixed with 4% formaldehyde (10 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 (ab7817, 0.1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113) at 1/2000 dilution for 30 min at 22°C

Isotype control antibody (black line) was mouse IgG2a [18C8BC7AD10] (ab170191) 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 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

Flow Cytometry (Intracellular) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)

Overlay histogram showing HeLa cells stained with ab29 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 20 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 (ab29, 0.1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 0.1μg/1x106 cells) used under the same conditions. 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.

Flow Cytometry - Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)

Overlay histogram showing HeLa cells stained with ab26 (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 (ab26, 1μg/1x10^6 cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10^6 cells) used under the same conditions. 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 HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)

ICC/IF image of ab7291 stained HeLa cells. The cells were 100% methanol fixed (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7291, 1μg/ml) overnight at +4°C. The secondary antibody (green) was ab150113 Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at 2μg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

The negative control (inset) is a secondary-only assay to demonstrate low non-specific binding of the secondary antibody.

Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 cells labelling Mouse IgG1 with ab280974 at 1/20 dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Red). The nuclear counterstain was DAPI (Blue).

Confocal image showing no staining in RAW 264.7 cells.

Negative control 1: ab280974 at a 1/20 dilution followed by ab150080 at a 1/1000 dilution.

Negative control 2: ab179513 at a 1/200 dilution followed by ab150113 at a 1/1000 dilution.

Alexa Fluor® - Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)