Human peripheral blood granulocytes stained with ab11867 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab11867, 1μg/1x106 cells) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - periphera
Immunohistochemical analysis of Human renal tissue, staining C5R1 with ab11867. For antigen retrieval, slides were immersed in citric acid buffer (pH 6) and were treated in a microwave oven for 10 minutes. Samples were blocked in 3% normal goat serum fr 30 minutes before incubating with primary antibody (1/50) overnight at 4°C. Staining was detected using DAB.
Immunohistochemical analysis of Human spleen (left) and resting neutrophils (right), staining C5R1 with ab11867. For antigen retrieval, slides were immersed in citric acid buffer (pH 6) and were treated in a microwave oven for 10 minutes. Samples were blocked in 3% normal goat serum fr 30 minutes before incubating with primary antibody (1/5) overnight at 4°C. A FITC-conjugated goat anti-mouse IgG (1/60) was used as the secondary antibody.