Programmed tissue arrayer

Formalin-fixed and paraffin-embedded tumour specimens, as well as fresh frozen tissue were obtained from the Institute of Pathology, University of Basel, Switzerland. For tissue microarray construction, a representative tumour area was selected on an H&E-stained slide of the donor block. Afterwards, a core punch with a diameter of 0.6 mm was taken from the tumour, and in selected cases from the non-tumoral liver tissue. Core punches were transferred to a new paraffin recipient block by using a programmed tissue arrayer (Beecher Instruments). The tissue microarray contained 30 HCC samples and 30 non-tumour liver control samples. Microarrays were cut in sections of 4 μm thickness and stained with a polyclonal LHPP antibody diluted at 1:200 using an automatic Benchmark XT staining machine (Ventana Medical Systems Inc.). LHPP staining intensity was evaluated by a clinical pathologist (M.S.M,) and graded semiquantitatively into: 0 for negative staining, 1 for weak positive staining, 2 for moderate positive staining and 3 for strong positive staining (Extended Data Fig. 6b). Ten pairs of samples in which one of the tissue sections was washed away during staining were ignored during evaluation. While scoring for staining intensity, the pathologist was double blinded to the antigen that was probed and the sample identity.