Immunocytochemistry - Anti-EGFR antibody [EP38Y] (ab52894) ab52894 staining of EGFR in a HCT116 cell spheroid. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.5% Triton X-100 for 1h and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween overnight at room temperature. The spheroids were then incubated overnight at room temperature with ab52894 at 2 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 2 µg/ml. DAPI was used as nuclear counterstain (shown in blue). As secondary antibodies ab150081 Goat anti-Rabbit (Alexa Fluor® 488) (shown in green) and ab150120 Goat anti-Mouse (Alexa Fluor® 594) (shown in magenta) were used, incubated overnight at room temperature. All permeabilization, blocking and antibody incubation steps were performed using a rotary shaker. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. The antibody ab52894 also worked using 100% methanol (5 min).

Western blot - Anti-EGFR antibody [EP38Y] (ab52894) All lanes : Anti-EGFR antibody [EP38Y] (ab52894) at 1000 µg Lane 1 : Wild-type A549 cell lysate Lane 2 : EGFR knockout A549 cell lysate Lane 3 : Wild-type HeLa cell lysate Lane 4 : EGFR knockout HeLa cell lysate Lysates/proteins at 20 µg per lane. Developed using the ECL technique. Performed under reducing conditions. Predicted band size: 134 kDa Observed band size: 175 kDa