The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
IHC-P: 1/50 - 1/100. Perform heat mediated antigen retrieval with 10 mM citrate buffer, pH 6.0 before commencing with IHC staining protocol.
WB: 1/500. Detects a band of approximately 23 kDa (predicted molecular weight: 23 kDa).
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
ab26249 (2µg/ml) staining CD3 epsilon in human spleen.
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 AR buffers citrate pH6.1. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min 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.