IHC image of CD5 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 ab238467 at 1µg/ml and ab16669 (Rabbit monoclonal [SP7] to CD3) at 1/150, to show the co-staining in 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.

Lewis rat splenocytes stained with ab238467 (right) or mouse IgG1κ (ab170190) 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 (ab238467) or mouse IgG1κ (ab170190) (1x¹⁰⁶ in 100 µl 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 on ice. The cells were simultaneously stained with CD3 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.