Alexa Fluor® 647 Anti-CD20 antibody [EP459Y]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-CD20 antibody [EP459Y] (AB198943)

Human peripheral blood lymphocytes stained with ab198943 (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. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab198943, 1/100 dilution) for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor^® 647 (ab199093) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >30,000 total events were collected using a solid-state 25mW red diode laser (635 nm) and 675/30 bandpass filter.

Gating strategy - lymphocytes.

• mIHC

Collaborator

Multiplex immunohistochemistry - Alexa Fluor® 647 Anti-CD20 antibody [EP459Y] (AB198943)

Immunofluorescence analysis of human rectum adenocarcinoma labelled with ab198943. Image was acquired using Cell DIVE Multiplexed Imaging Solution (Leica Microsystems).

Formalin fixed paraffin embedded (FFPE) sections were incubated with ab198943 at 1/100 dilution, for 1h at room temperature. Nuclear DNA was labelled with DAPI (Red). Deparaffinization, antigen retrieval, biomarker labeling and imaging was performed following Cell DIVE recommended standard protocol. Cell DIVE automatically processed the image to perform autofluorescence removal, distortion correction, blank glass subtraction, flat-field correction, and stitching.