IHC detection
IHC was used to detect the in situ expression of EBI3 and IL12A
in HCC tumor and peri-tumor samples. After paraffin-embedded tissue
sections were deparaffinized with xylene and graded alcohols,
heat-activated antigen retrieval was performed in EDTA antigen retrieval
solution (Beyotime, Shanghai China). A standard two-step protocol was
applied with the following primary antibodies incubated overnight at 4
°C: rabbit anti-human EBI3 polyclonal antibody (1:100, ab8396; Abcam,
Cambridge, MA, USA) and rabbit anti-human IL12A monoclonal antibody
(1:1000, ab131039; Abcam) Then, the tissue sections were serially rinsed
and antibody binding was detected using a peroxidase-conjugated
secondary antibody (Absin, Shanghai, China) for 30 min at room
temperature. Finally, slides were counterstained with hematoxylin,
dehydrated in ethanol, cleared in xylene and covered with slips. IHC
slides were scanned using an Automatic Digital Slice Scanning
System(PRECICE 500, Beijing, China).