Dab horseradish peroxidase color development kit

All of the 120 patient tumor tissue samples were analyzed immunohistochemically. Immunohistochemistry (IHC) was performed on 5 mm sections of formalin-fixed, paraffin-embedded tissue samples. The paraffin sections were deparaffinized with xylene and rehydrated in alcohol. Antigen retrieval was accomplished by boiling citrate buffer and endogenous peroxidase activity was blocked with 3% H₂O₂ followed by staining with anti-PD-L1 antibody (1:100; Cell Signaling Technology, Danvers, MA, USA), anti-CD4 antibody (1:500; ZSGB-BIO, Beijing, China) or anti-CD8 antibody (1:500; ZSGB-BIO, Beijing, China) overnight at 4 °C. After washing, the sections were processed with a MaxVision^TM HRP-Polymer anti-Rabbit IHC Kit at room temperature (Maixin, Fuzhou, China) and then developed with a DAB Horseradish Peroxidase Color Development Kit (Maixin, Fuzhou, China) and counterstained with hematoxylin.

This procedure was performed as previously described [16 (link), 17 (link)]. In short, paraffin sections were deparaffinized using xylene and rehydrated using a series of alcohol solutions. Endogenous peroxidase activity was blocked by treatment with 3% H₂O₂ for 15 min. Then the sections were incubated with an anti-ZFP36L1 (Abcam, 1:100) antibody overnight at 4 °C. After washing with PBS three times, the tissue slides were treated with secondary antibody using a MaxVision™ HRP-Polymer anti-Rabbit IHC Kit (Maixin, China). The color was developed using a DAB Horseradish Peroxidase Color Development Kit (Maixin, China), and the sections were counterstained with haematoxylin. The degree of immunostaining was scored according to both the proportion of positively stained tumor cells and the staining intensity. The score for each tissue was calculated by multiplying the staining index (0, 1, 2 and 3) by the percentage category value (0, 1, 2, 3 and 4), and the average of the scores from the two pathologists was used as the final score.