Horseradish peroxidase polymer

For IHC, tumor tissues were extracted, fixed for 24 h in 10% formaldehyde, and embedded in paraffin. IHC staining was performed as follows. Tissue sections were deparaffinized twice with xylene for 10 min and were rehydrated using a graded alcohol series. After removing endogenous peroxidases using 0.1% H₂O₂, sections were washed three times with PBS. Antigen retrieval was achieved by incubating the sections in 10 mM citrate buffer (pH 6.0) (DAKO, Glostrup, Denmark) using a microwave oven. Then the sections were permeabilized with 0.5% PBX (0.5% Triton X-100 in PBS) for 30 min and washed three times with PBS. After blocking for 1 h with 5% BSA, the primary antibody was added, and the sections were incubated overnight at 4 °C. Primary Antibody Enhancer (Thermo Fisher Scientific, Waltham, MA, USA) and horseradish peroxidase Polymer (Thermo Scientific) were used for signal amplification. To develop the colored product, a mixture of DAB (3,3′-diaminobenzidine) Plus Chromogen and DAB Plus Substrate (Thermo Fisher Scientific) was added for 5 min. After washing with PBS, 20% hematoxylin counterstain was added for 2–5 min to stain the nuclei. Finally, the tissue sections were dehydrated in a graded alcohol series. After clearing twice in xylene, the tissue sections were coverslipped with mounting media (xylene:mount = 1:1) for microscopy.

For IHC, tumor tissues of two mice per group after 7 days of third virus injection were extracted after CO₂ euthanasia. Then, they were fixed for 24 h in 10% formaldehyde, and embedded in paraffin. Tissue sections were deparaffinized twice with xylene for 10 min and were rehydrated using a graded alcohol series. After antigen retrieval, the sections were permeabilized with 0.5% PBX (0.5% Triton X-100 in PBS) for 30 min and washed three times with PBS. After blocking for 1 h with 5% BSA, the sections with primary antibodies were incubated overnight at 4 °C. Primary Antibody Enhancer (Thermo Fisher Scientific) and horseradish peroxidase Polymer (Thermo Scientific) were used for signal amplification. To develop the colored product, a mixture of DAB (3,3′-diaminobenzidine) Plus Chromogen and DAB Plus Substrate (Thermo Fisher Scientific) was added for 5 min. After washing with PBS, 20% hematoxylin counterstain was added for 2–5 min to stain the nuclei. After dehydration and clearing twice in xylene, the tissue sections were coverslipped with mounting media (xylene:mount = 1:1) for microscopy.