ab107846 stained JEG3 cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab107846 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - Anti-ITIH5 antibody (ab107846)
Anti-ITIH5 antibody (ab107846) at 1/100 dilution + NCI-H460 cell line lysate at 35 µg
Secondary HRP conjugated anti-rabbit at 1/10000 dilution
Immunohistochemistry analysis in formalin fixed and paraffin embedded Human breast carcinoma followed by peroxidase conjugation of the secondary antibody and DAB staining. ab107846 at a dilution of 1/50.