ICC/IF image of ab43057 stained PC12 cells. The cells were 100% methanol fixed (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 (ab43057, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Ab43057 staining human normal placenta tissue. Staining is localised to cytoplasm.Left panel: with primary antibody at 1 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 AR buffer citrate pH6.1 in a DAKO PT Link. . 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.
All lanes : Anti-14-3-3 epsilon antibody - Aminoterminal end (ab43057) at 1/500 dilutionLane 1 : Brain (Human) Tissue Lysate - adult normal tissue (ab29466)Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell LysateLane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.