Immunohistochemical Analysis of PD-L1 in Mouse Tumors

Mouse tumor paraffin sections were de-paraffinized and rehydrated. Antigen retrieval was performed in 10 mM citrate buffer pH 6.0 for 10 min with a pressure cooker followed by blocking endogenous peroxidase with 3% H₂0₂ for 15 min. Blocking was also performed for endogenous biotin with an avidin-biotin blocking kit. Samples were incubated with 10% goat serum overnight at 4°C. The next day, sections were stained with anti–PD-L1 rabbit primary antibody and incubated with a biotinylated goat-anti-rabbit IgG antibody followed by an avidin-biotinylated horseradish peroxidase complex. Immune-complexes were visualized using the Opti-View DAB IHC Detection Kit followed by an Opti-View Amplification Kit. Immunoreactivity and samples were counterstained with hematoxylin.