Immunohistochemical Analysis of UBE2T

A paraffin slicing machine (Leica, Germany) was used to cut paraffin-embedded samples into 4 μm slices. IHC staining was performed following the standard protocol as previously described [18 (link)]. After initial deparaffinization, antigen retrieval, and blockade, the anti-UBE2T antibody (cat#: ab154022, Abcam, Cambridge, MA, United States) was applied to sections overnight at 4 °C in a humidified container. A horseradish peroxidase-labeled secondary antibody (cat#: ab205718, Abcam, Cambridge, MA, United States) followed the next day. DAB (50:1, Novus Biologicals, Centennial, CO, United States) was used to visualize UBE2T staining, followed by hematoxylin counterstain. Sections skipping the primary antibody were used as the negative control. Each sample was designated a score of 0 (negative), 1 (mild), 2 (moderate), or 3 (strong) based on the relative staining intensity. Tissue with score ≤ 1 or ≥ 2 was defined as low and high expression, respectively.