Anti-PD1 antibody [NAT105] (ab52587)

IHC image of PD1 staining in normal human tonsil formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab52587 at 5 µg/ml for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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

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

Formaldehyde-fixed, paraffin-embedded Xhuman follicular lymphoma tissue stained for PD1 with ab52587 at 1/100 dilution in immunohistochemical analysis.

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MOLT4 cells stained for PD-1 (colored green) using ab52587 in ICC/IF. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Tween-20 for 1 hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the PD-1 antibody ab52587 at 10 µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) at 1 ug/ml overnight at +4°C. The secondary antibodies used were Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150117) secondary antibody used at 1 ug/ml (colored green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) (ab150080) secondary antibody (pseudo-colored red) used at 2 ug/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

Overlay histogram showing MOLT4 cells stained with ab52587 (red line). Live cells were incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab52587, 1/100 dilution) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150117) at 1/2000 dilution for 30 min at 4°C.

Immunohistochemical analysis of human large and locally advanced breast cancers staining PD-1 using ab52587. (a) Low level of PD-1⁺ T cell infiltration; (b) high level of PD-1⁺ T cell infiltration. (Itu: intratumoral Str: stromal)

Immunohistochemical analysis of frozen Human liver tissue labeling PD1 with ab52587 at 1/50 dilution.

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Immunohistochemical analysis of various soft-tissue sarcomas staining PD1 using ab52587. Arrows indicate PD1 positive lymphocytes.