ab170190) isotype (left). Cells 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 (ab243838) or mouse IgG1κ (ab170190) isotype (1x 10^{ab150117) was used at 1/2000 dilution for 30 min on ice.}

IHC image of CD200R 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 ab243838 (shown in green) at 5μg/ml and co-localisation of T cells stained using ab16669 (shown in red), Rabbit monoclonal to CD3, at 1/150 dilution. The section was then incubated with ab150117 (Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 488, 1/1000)) presabsorbed and ab150080 (Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594, 1/1000)) for 1 hour at room temperature. The secondary-only control 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.