ab51317 stained mouse TH2 cells. The cells were 4% formaldehyde fixed for 4 minutes at room temperature 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 (ab51317 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (ab150165) used at a 1/1000 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.

Flow cytometry staining of C57 BL/6 mouse splenocytes with ab51317 (right) or Rat IgG2aκ (ab18450) isotype (left). Cells were incubated for 30 min on ice in 1x PBS containing 10 % mouse serum/10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab51317) or Rat IgG2aκ (ab18450) isotype (1x 10⁶ in 100μl at 0.2μg/ml) for 30 min on ice.

The secondary antibody Goat anti-rat IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150165) was used at 1/2000 dilution for 30 min on ice. The cells were simultaneously stained with CD4.

Acquisition of >30,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were gated on live lymphocytes.