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

5 product images

• 

  Flow Cytometry - Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488) (ab150177)

  Overlay histogram showing HeLa cells stained with Anti-Alpha-synuclein antibody 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 (Anti-Alpha-synuclein 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 (Sheep IgG - Isotype Control 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.

• 

  Immunocytochemistry/ Immunofluorescence - Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488) (ab150177)

  ICC/IF image of Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, 5µg/ml) overnight at +4°C. Sheep Anti-Mouse IgG H&L 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.

• 

  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® - Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488) (ab150177)
• 

  Immunocytochemistry/ Immunofluorescence - Donkey Anti-Sheep IgG H&L (Alexa Fluor® 488) (ab150177)

  Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker ab92539 stained in SHSY5Y cells. Cells were fixed with 100% methanol (5 min), permeabilized 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 the antibody Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker ab92539 at 0.5µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed ab150087 (colored red) and ab150177 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  This product gave a positive signal in 4% formaldehyde (10 min) fixed SHSY5Y cells under the same testing conditions.

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