ICC/IF image of beta Tubulin staining in 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 primary antibody (ab6046, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab150077 Alexa Fluor^® 488 goat anti-rabbit 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 analysis of C6(Rat glial tumor glial cell) cells labeling alpha smooth muscle Actin with purified ab32575 at 1/100 dilution (0.71 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Overlay histogram showing MCF7 cells stained with unpurified ab32063 (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 goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32063, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (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. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3(Mouse embryonic fibroblast) cells labeling alpha smooth muscle Actin with purified ab32575 at 1/500 dilution (5.2 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling Estrogen Receptor alpha with purified ab32063 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue).
Control 1: primary antibody (1/1000) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).
Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

Immunocytochemistry/Immunofluorescence analysis of Raji (human Burkitt's lymphoma) cells labelling HLA A with purified ab52922 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.
Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).
Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

Overlay histogram showing Jurkat cells stained with ab16669 (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 goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab16669, 1/1000 dilution) for 30 min at 22°C. The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077) was used at 1/4000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monclonal) (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 analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling ATG9A with Purified ab108338 at 1/100 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG (Alexa Fluor® 488) was used as the secondary antibody at 1/1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunocytochemistry/Immunofluorescence analysis of A431 (human epidermoid carcinoma) cells labeling alpha smooth muscle Actin (green) with purified ab32575 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counterstained with ab7291, anti-Tubulin (mouse mAb) at 1/1000 followed by ab150120 Alexa Fluor®594 goat anti-mouse secondary (1/1000). Nuclei were counterstained with DAPI (blue).
For negative control 1, rabbit primary antibody and anti-mouse secondary antibody (ab150120) were used. For negative control 2, ab7291 (mouse primary antibody) was used followed by anti-rabbit secondary antibody (ab150077).

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling IL-1RA with purified ab124962 at 1/100. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.
Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).
Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 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, 1µg/ml) and (ab16048, 1µg/ml) overnight at +4°C. The secondary antibodies were ab150115 Alexa Fluor® 647 (red) goat anti-mouse IgG (H+L) used at 2µg/ml for 1h and ab150077 Alexa Fluor® 488 (green) goat anti-rabbit IgG (H+L) used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei.

Overlay histogram showing HeLa cells stained with ab32356 (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 (ab32356, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/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.

Cross-reactivity of the polyclonal secondary antibody ab182016 was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards at 1 µg/ml (50 µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. ab182016 was then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Donkey anti-Goat IgG H&L (HRP) (ab6885) was used at 1/10,000 dilution (50 µl/well), followed by incubation for 1h at RT.

For the batch tested, ab182016 showed a cross-reactivity of 5-7% towards Human IgG and below 2% towards Mouse IgG, Rat IgG and Chicken IgY.

This data was developed using the unconjugated antibody (ab182016).

Cross-reactivity of Goat anti-Rabbit IgG H&L (ab182016) and Goat anti-Rabbit IgG H&L obtained from two different vendors was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards (Rabbit, Human, Mouse and Rat) at 1 µg/ml (50 µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. Secondary antibodies were then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Donkey anti-Goat IgG H&L (HRP) (ab6885) was used at 1/10,000 dilution (50 µl/well), followed by incubation for 1h at RT. This data is from a representative dilution.

This data was developed using the unconjugated antibody (ab182016).

IHC - Wholemount of Caenorhabditis elegans larvae labelling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab5095. The sample was incubated with primary antibody (1/500 in PBS + 3% BSA + 0.1% Triton X-100) for 12 hours at 4°C. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal (1/1000), was used as the secondary antibody.

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