All lanes : Anti-E2F1 (phospho T433) antibody (ab55325) at 1/500 dilutionLane 1 : HeLa cell extract treated with Etoposide (at 25µM for 24 hrs).Lane 2 : HeLa cell extract treated with Etoposide (at 25µM for 24 hrs), and with the immunising phosphopeptide.
Ab55325 staining human normal pancreas. Staining is localised to nuclear compartment.Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control.Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer citrate pH6 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.