IHC image of CD62P staining in a section of frozen normal human tonsil*. 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 ab54427 at 5µg/ml. The section was then incubated with ab150117 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488), 1/1000)) (shown in green) for 1 hour at room temperature. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. The secondary-only control insert image is taken from an identical assay without primary antibody. 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. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

Flow cytometry staining of  human whole blood with ab54427 (right) or mouse IgG1κ (ab170190) isotype (left). Red blood cells of 200 µL blood were lysed, then cells were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10% normal goat serum to block Fc receptors and non-specific protein-protein interaction followed by the antibody (ab54427) or mouse IgG1κ (ab170190) isotype (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 CD42a PE.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on alive neutrophils.