C57BL6 mouse splenocytes were incubated with Rat IgG2b, kappa monoclonal [RTK4530] (ab18541) (left) or anti-P2X7 [F11] antibody (ab195356) (right) at 10 µg/mL for 30 min at +4°C. The secondary antibody Goat Anti-Rat IgG H&L (Alexa Fluor® 488) (ab150157) was used at 1/2000 dilution for 30 min at +4°C. The cells were simultaneously stained with anti-CD3ε PerCP (10 µg/mL) (ab190299).

Acquisition of >30,000 total events were collected. Gating strategy - live lymphocytes

IHC image of ab195356 staining in 10% formaldehyde fixed frozen tissue section of normal mouse large intestine.

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 1h at room temperature. The section was then incubated with ab195356 (1μg/ml) 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)) and DAPI for 1 hour at room temperature.

The DAPI only control (no antibody) inset shows no autofluorescence, demonstrating that any Alexa Fluor^® 647 signal is derived directly from bound ab195356. 

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