Overlay histogram showing peripheral blood lymphocytes stained with ab6124 (red line). The cells were incubated with the antibody (ab6124, 0.5µg/1x10⁶ cells) for 30 min at 4ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H&L) (ab96879) at 1/200 dilution for 30 min at 4ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed gating on peripheral blood lymphocytes.

Immunohistochemical analysis of Human esophogeal keratinocytes, staining CD44 with ab6124.

Keratinocytes were cultured in vivo to form an epithelium for 15 days before fixing in formaldehyde and embedding in paraffin. Primary antibody was incubated overnight at 4°C and secondary antibody for 30 minutes at 37°C. Staining was detected using DAB.

ab6124 staining CD44 in A431 cells. The cells were fixed with 100% methanol (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 ab6142 at 5µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

IHC image of CD44 staining in human kidney carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab6124, 5µg/ml, for 15 mins 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.