Lewis rat splenocytes stained with ab256254 (right) or mouse IgG1κ Alexa Fluor^® 488 isotype (left). Lewis rat splenocytes were incubated for 30 min on ice in 1x PBS / 10 % rat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab256254) or mouse IgG1κ Alexa Fluor^® 488 isotype (1x 10⁶ in 100 µl at 0.2 µg/ml (1/2500)) for 30 min on ice. The cells were simultaneously stained with CD3.

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.

IHC image of CD45 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 (w/v) 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 ab256254 at 1/50 dilution (shown in green). Nuclear DNA was labeled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

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

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