Envision system horseradish peroxidase hrp labeled polymer anti mouse

Serial sections of three-micrometer-thick formalin-fixed, paraffin-embedded tissue sections were handled as described (7 (link)). Incubation conditions for the primary antibodies are included in Supplementary Table 1. EnVision™ system-horseradish peroxidase (HRP)-labeled polymer anti-mouse (Dako, ref. K4000) for mouse primary antibodies and EnVision™ system-HRP-labeled polymer anti-rabbit (Dako, ref. K4011) for rabbit primary antibodies were incubated for 40 min at room temperature.
For the negative controls, the same protocols were followed, but the primary antibody was omitted to avoid non-specific signals.

Immunohistochemical staining for Trop-2 was performed using 4 μm-thick paraffin-embedded tissue sections from the UTUC-TMA, which were deparaffinized using xylene and a graded series of ethanol concentrations. For Trop-2 antigen retrieval, the sections were treated with Tris-ethylenediaminetetraacetic acid (EDTA) buffer (pH 7.0) and steamed by placing them above boiling water for 20 min. Endogenous peroxidase activity was blocked by incubating the sections with 0.3% hydrogen peroxide for 5 min, followed by overnight incubation with primary antibodies against Trop-2 (1:200; SC-376181, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C.
Then, we used the EnVision + system-horseradish peroxidase (HRP)-labeled polymer anti-mouse (DAKO) according to the manufacturer’s instructions. Sections were counterstained with hematoxylin, dehydrated using a graded series of ethanol concentrations, cleared in xylene, and mounted for viewing under a microscope. The intensity and extent of Trop-2 expression were determined using the histochemical scoring system (H-score), which was based on the product of the staining intensity (score, 0–3+) and percentage of stained cells (0–100%) at a given intensity. Specimens were subsequently classified as negative (0+, H-score 0–14), low (1+, H-score 15–99), moderate (2+, H-score 100–199), and high (3+; H-score, 200–300).