Tissue Microarray Preparation and IHC Analysis

Tissue Array Preparation: We followed the methods described by Wang et al.^{12 (link)}. To summarize, the Quick-Ray® UT-06 tissue microarray system and the Quick-Ray premade recipient block (UB-06) wax model, both produced by Unitma Co., Ltd. in Seoul, Korea, were utilized for the preparation of tissue specimens measuring 1 mm in diameter. Two specific locations were chosen from each sample of liver cancer tissue for sampling purposes. IHC Analysis: The Envision System (Dako, Glostrup, Denmark) was used for IHC staining of paraffin-embedded tissue sections, following the method previously described^{13 (link),14 (link)}. Primary antibodies used in the experiment included the anti-FSCN1 mouse monoclonal antibody (clone 55 k-2; diluted to a concentration of 1:100; obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA). For the secondary antibody, Dako's HRP rabbit/mouse universal antibody (from Dako, Glostrup, Denmark) was employed. In the negative control, the vehicle was initially incubated, followed by the secondary antibody, without any primary antibody. As positive control, we used the internal control in the liver tissue, to check for FSCN1 staining in the interstitial fibers and vascular endothelial cells.