Polyperoxidase conjugated igg

Tissue microarray (TMA) construction and the immunohistochemistry protocol were conducted as previously described.¹⁷ For each patient, three sections of tumor tissue were taken from paraffin blocks to construct TMA. Primary antibodies (listed in supplementary table 2) were applied to sections at 4°C overnight for immunohistochemical staining. For different T helper cells, TH1 was defined as CD4⁺ T-bet⁺ cells and TH2 was defined as CD4⁺ GATA3⁺ cells. Then, slides were treated with polyperoxidase-conjugated IgG (OriGene). Staining was performed with diaminobenzidine solution (Biocare Medical) under a microscope and counterstained with hematoxylin. The primary antibody was omitted for the negative controls. The positive cells were enumerated from the representative view of the three sections in high-power field (HPF) and an average number was adopted. Two pathologists who were unaware of patient information evaluated the staining of each specimen with the assistance of Image-Pro Plus 6.0 (Media Cybernetics Inc.). In case of disagreement, the slides were reviewed and the two observers achieved a consensus.