Lewis rat splenocytes stained with ab238473 (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 (ab238473) 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.

IHC image of CD62L 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 ab238473 at 1µg/ml and ab16669 (Rabbit monoclonal [SP7] to CD3) at 1/150, to show the co-staining in naive T cells. The section was then incubated with ab150117 (Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preabsorbed, (Shown in green) 1/1000) and ab150080 (Goat Anti-Rabbit IgG H&L (Alexa Fluor^®594) (Shown in red) 1/1000) for 1 hour at room temperature. The secondary-only control insert image is taken from an identical assay without primary antibody. DAPI was used to stain the cell nuclei (blue). 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.