ICC/IF image of ab6160 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 100% methanol 5 minutes, 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 1 hour to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6160, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was ab150165 Alexa Fluor^® 488 goat anti-rat IgG (H+L) pre-adsorbed, used at a 1/1000 dilution for 1 hour. 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.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine and sodium azide (ab6160).

IHC image of Tubulin staining in human colon formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer pH 6 for 20 minutes. The section was then incubated with ab6160, 5 µg/ml, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine and sodium azide (ab6160).

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with ab6160 (red line).

The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. 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 (ab6160, 1 µg/1x10⁶ cells) for 30 minutes at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was rat IgG2a [aRTK2758] (ab18450, 1 µg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine and sodium azide (ab6160).

This image was kindly supplied as part of the review submitted by Marko Kallio. ab6160 was used for immunofluorescence on male rat testis samples in order to visualize microtubules of meiotically deviding cells. The samples were fixed with 2% paraformaldehyde and 0.8% glutaraldehyde and the antibody was used at a dilution 1:2500 (red - tubulin, blue - DNA stained with DAPI).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine and sodium azide (ab6160).