Vectra inform image analysis system

To assess the expression level of GLUT1/ASCT2, the Vectra-Inform image analysis system (Perkin-Elmer Applied Biosystems) was used as described in previous studies [18 (link), 19 (link)]. In brief, a scanning protocol was designed on the basis of the TMA core number and size. Nuance multispectral image cubes (8-bit) were acquired from the TMA at a constant 200× magnification. The spectral library containing two chromogens was built with two control HCC tissue slides stained with only 1 chromogen (3,3′-diaminobenzidine or hematoxylin). The spectral library was used to unmix the signals on the multicolored images by recognizing their unique spectral curves for quantitation. Images were analyzed using InForm image analysis software (version 2.0.1; Perkin-Elmer Applied Biosystems) to identify tissue compartments (tumor and non-tumor tissue) and subcellular compartments (nucleus, cytoplasm, and membrane). The H-score was calculated by quantifying target signals in selected tissues and cellular compartments of interest following the manufacturer’s protocol.

Immunohistochemical (IHC) staining was performed as described in previous study [16 (link)]. Briefly, Tissue sections prepared for antigen retrieval by microwave treatment in citrate buffer (pH 6.0) were incubated with anti-NAP1L1 (Sigma, America), anti-Ki67 (Zsbio, China), anti-cleaved-caspase 3 (Affinity, China) primary antibodies. Immunostaining was performed using the Envision System with diaminobenzidine (Dako Cytomation, Glostrup, Denmark). To assess the expression level of NAP1L1 in HCC tissue microarrays, a Vectra-Inform image analysis system (Perkin-Elmer Applied Biosystems) was used as described in previous studies [17 (link), 18 (link)].

Paraffin-embedded and formalin-fixed HCC samples were cut into 5-μm sections, which were then processed for immunohistochemistry as previously described. Briefly, the sections were incubated with Abs against the following human antigens: CD8 (rabbit monoclonal, clone EP334, ZSBio, China), CD11b (rabbit monoclonal, clone EPR1344, Abcam, UK), CD68 (mouse monoclonal; clone PG-M1; Dako Cytomation, USA), CD15 (mouse monoclonal; clone LeuM1; ZSBio), CD206 (mouse monoclonal, clone 685645, R&D Systems, USA), CD204 (mouse monoclonal, clone SRA-C6, Transgenic, Japan), CD163 (mouse monoclonal, clone 10D6, ZSBio) and CD169 (sheep polyclonal, clone NS0, R&D Systems). Then, the sections were stained in an EnVision System (Dako Cytomation, USA). Positive cells were detected by microscopy and quantified using the Vectra-Inform image analysis system (Perkin-Elmer Applied Biosystems, Foster City, CA, USA).