IHC image of CD43 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 ab22351, 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.

*Tissue obtained from Charles River.

IHC image of CD43 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 ab22351 (shown in red) at 1µg/ml and co-localization of T cells stained using ab16669 (shown in green), Rabbit monoclonal to CD3, at 1/150 dilution. The section was then incubated with ab150119 (Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^®647, 1/1000)) and ab150077 (Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^®488, 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.

Lewis rat splenocytes stained with ab22351 (right) or mouse IgG1κ (left). Lewis rat splenocytes were incubated for 30 min on ice in 10% rat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab22351) or mouse IgG1κ Isotype (ab170190) (1x10⁶ in 100µl 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 at 4°C. The cells were simultaneously stained with CD3 APC antibody.

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