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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and Sodium Azide (ab33945)