Recombinant Alexa Fluor® 647 Anti-PD1 antibody [NAT105] (ab279695)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab279695 (right) or mouse IgG1κ Alexa Fluor^® 647 isotype (left). PBMCs were incubated for 30 min on ice in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab279695) or mouse IgG1κ Alexa Fluor^® 647 isotype (100 μl at 1μg/ml (1/500)) for 30 min on ice. The cells were simultaneously stained with CD3.

Acquisition of >30000 events were collected using a 40 mW Red laser (638nm) and 660/10 bandpass filter. Events were gated on live lymphocytes.

Immunofluorescence staining of PD1 staining in a section of formalin-fixed paraffin-embedded human normal tonsil*.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Biocare Medical NxGen pressure cooker using retrieval settings of 110°C for 20 minutes. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) 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 ab279695 at 1/100 dilution (shown in red) and counterstained using ab195887, Ms anti-Tubulin (A488) (1/250) at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

Immunohistochemical analysis of formalin-fixed paraffin-embedded human normal tonsil* tissue labelling PD1.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Biocare Medical NxGen pressure cooker using retrieval settings of 110°C for 20 minutes. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) 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 ab279695 at 1/100 dilution (shown in red) and counterstained using ab195887, Ms anti-Tubulin (A488) (1/250) at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.