Immunohistochemical Analysis of URI1 in HCC

Patients with HCC are summarized in Supplementary Table S2. Formalin-fixed paraffin-embedded tissue specimens were cut into 4 μm sections. After dewaxing in xylene, tissue sections were autoclaved for 10 min in a citrate buffer, pre-incubated in 0.3% H₂O₂, and blocked for 20 min using diluted normal blocking serum from a VECTASTAIN Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA). Sections were incubated with 1:100 diluted anti-URI1 antibody as a primary antibody followed by an HRP-conjugated secondary antibody (Vector Laboratories). URI1 protein was visualized using ImmPACT DAB substrate (Vector Laboratories) with a reaction time of 3 min.
Paired HCC-B and non-tumor liver tissues from five patients (Supplementary Table S3) were used for the ChIP assay, which was performed as previously reported [41 (link)].
These studies were approved by the ethical committee of Tottori University (18A071).