All lanes : Anti-eIF4B (phospho S422) antibody (ab59300) at 1/500 dilutionLane 1 : extracts from NIH/3T3cellsLane 2 : extracts from NIH/3T3cells with immunizing peptide
Ab59300 at 1/50 dilution staining human colon carcinoma without (left) and with (right) immunizing peptide; paraffin embedded.
ICC/IF image of ab59300 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab59300, 1µg/ml) 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.