Flow cytometry staining of Lewis rat splenocytes with ab33945 (right) or mouse IgG1&kappa (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 (ab33945) or mouse IgG1&kappa (ab170190) isotype (1x10⁶ in 100 μl; at 0.2 μg/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.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were gated on live single cells.

IHC image of CD45RC 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 blocked using TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1 hour at room temperature. The section was then incubated with ab33945 (1µg/ml dilution) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. The section was then incubated with ab150119 ((Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647) (shown in red) and DAPI (staining nuclear DNA) (shown in blue) for 1 hour at room temperature. The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

The DAPI only control (no antibody) inset shows no autofluorescence, demonstrating that any Alexa Fluor^® 647 signal is derived directly from bound ab33945.

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

*Tissue obtained from Charles River.

IHC image of CD45RC staining in a section of formalin-fixed paraffin-embedded normal rat spleen performed on a Leica BOND™ system using the standard Protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab33945, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX.

The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times