Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488)

• Flow Cyt

Lab

Flow Cytometry - Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488) (AB150177)

Flow cytometry overlay histogram showing left Jurkat positive cells and right negative HeLa stained with ab230296 (red line). The cells were incubated in 1x PBS containing 10% human IgG and 10%; normal goat serum to block non-specific protein-protein interaction followed by the antibody ab230296 at 1 μg/ml for 30min on ice.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150177) was used at 1/4000 dilution for 30 min at 4°C. Isotype control antibody (black line) was mouse IgG2a (ab18413) used at the same concentration and conditions as the primary antibody. Unlabeled 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. Events were gated on viable single cells.

• Flow Cyt

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Flow Cytometry - Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488) (AB150177)

Overlay histogram showing HeLa cells stained with ab21976 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal donkey serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab21976, 0.1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody Donkey anti-sheep IgG H&L (Alexa Fluor^® 488) (ab150177) was used at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was sheep IgG (ab37385, 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488) (AB150177)

ICC/IF image of ab7291 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7291, 5µg/ml) overnight at +4°C. ab6710, sheep anti-mouse IgG, was then added as a secondary bridging antibody, at 1/250 dilution for 1h. ab150177 Alexa Fluor® 488 donkey anti-sheep IgG (H+L) (green) was then used at 2µg/ml for 1h as a tertiary antibody. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488) (AB150177)

HeLa cells showing negative staining by ICC/IF using only secondary antibody. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. ab150177 Alexa Fluor® 488 donkey anti-sheep IgG (H+L) (green) was used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• Alexa Fluor®

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Alexa Fluor® - Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488) (AB150177)