Lewis rat splenocytes (top) or Lewis rat splenocytes treated with 5µg/ml ConA for 3 days (bottom) were stained with ab238475 (right) or mouse IgG2b kappa (ab170192) isotype (left). Splenocytes were incubated for 30 min on ice in 1x PBS containing 10 % rat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab238475) or mouse IgG2b kappa (ab170192) isotype (1x 10⁶ in 100µl at 0.2µg/ml) for 30 min on ice.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^®488, pre-adsorbed) (ab150117) 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 CD3 positive T cells.

IHC image of CD134/OX40L receptor staining in a section of frozen normal rat spleen*.

The section was fixed using 10% formaldehyde in 1XPBS for 10 minutes. No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M glycine and 1% (w/v) BSA for 1h at room temperature.  The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab238475 at 5µg/ml. The section was then incubated with ab150119 (Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647, 1/1000)) (shown in red) for 1 hour at room temperature. The secondary-only control insert image is taken from an identical assay without primary antibody. The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). For IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

*Tissue obtained from Charles River.