C57BL/6 mouse splenocytes stained with ab235126 (right) or rat IgG2aκ (left). C57BL/6 Mouse splenocytes were incubated for 30 min on ice in 10% mouse serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab235126) or rat IgG2aκ Isotype (ab18450) (1x10⁶ in 100µl at 0.1 µg/ml) for 30 min on ice.

The secondary antibody Goat anti-rat IgG H&L (Alexa Fluor ^® 488, pre-adsorbed) (ab150165) was used at 1/2000 dilution for 30 min at 4°C.The cells were simultaneously stained with CD19 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.

IHC image of IgD staining in a section of frozen normal mouse 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 ab235126 (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 ab150167 (Goat polyclonal Secondary Antibody to Rat IgG - H&L (Alexa Fluor^® 647) preabsorbed) (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 ab235126.

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.