Horseradish peroxidase hrp labeled goat anti mouse rabbit secondary antibody

Standard immunohistochemical analysis was performed with the primary antibody against human PD-1 (clone NAT105, mouse immunoglobulin G1, Abcam, Cambridge, UK) and PD-L1 (clone SAB2900365, rabbit immunoglobulin G1, Sigma-Aldrich, Saint Louis, USA) at a dilution in 1:100 and 1:400, respectively. Briefly, antigen retrieval was achieved by microwave pretreatment in citrate buffer. After neutralization of endogenous peroxidase, tissue microarray slides were preincubated with blocking serum and then were incubated with PD-1 or PD-L1 antibody for 40 minutes at room temperature. After three washes in PBS, the slides were treated with the horseradish peroxidase (HRP)-labeled goat anti-mouse/rabbit secondary antibody (Dako, Glostrup, Denmark) for 20 minutes at room temperature, then continued to wash in PBS. Finally, reaction products were visualized with 3,3′-diaminobenzidine (DAB, Dako, Glostrup, Denmark) and the slides were counterstained with hematoxylin. After being dehydrated, slides were mounted in resin.

The IHC Envision staining method was used for immunohistochemical staining. Antigen retrieval was performed by pressure oven 2.5 min in EDTA (pH 9.0). Slides were incubated for 15 min with H₂O₂ (Dako, Glostrup, Denmark). The primary antibody, rabbit anti-PD-L1 monoclonal antibody (clone SP142; 1:50, Spring Bioscience, Pleasanton, CA, USA) was incubated sequentially for two hours at room temperature. The bound antibody was then detected with the horseradish peroxidase (HRP)-labeled goat anti-mouse/rabbit secondary antibody (Dako, Glostrup, Denmark) for 30 min at room temperature. Finally, reaction products were visualized with 3, 3′-diaminobenzidine (DAB, Dako, Glostrup, Denmark). Slides were lightly counterstained with hematoxylin.