Vectra polaris multispectral imaging system

A total of 235 formalin‐fixed paraffin‐embedded liver cancer samples were collected randomly from HCC patients at Zhongshan Hospital, Fudan University (Shanghai, China) during 2006 and 2007. As mentioned previously, tissue microarrays (TMAs) were conducted by Shanghai Biochip Co., Ltd. It was carried out according to the instruction by the multiplex quantitative immunofluorescence staining for TMAs slides. Slides were fluorescently stained with CD8, PD1, CD4, and FOXP3 antibodies by Opal 7‐Color Manual IHC Kit (NEL811001KT), and the Vectra Polaris multispectral imaging system (PerkinElmer) was used to acquire multispectral images of arrays. As reported before,²⁶ after multilabeled immunofluorescence, cells were identified through the Cell Segmentation function of Inform software. Then Score function was used to calculate fluorescence intensity. A positive rate of cells refers to the fluorescence intensity of a single‐cell exceeded the threshold value. Positive rates of makers (such as CD8⁺, CD8⁺PD‐1⁺) in peritumor or tumor were divided into low and high expression groups according to a cutoff value of overall survival (OS) assessed by X‐tile 3.5.0.

Mouse tissues were dehydrated after being fixed for minimum 48 h in 4% PFA at 4 °C. The tissues were after subsequently embedded in OCT compound (Sakura-Finetek, CA, USA) and frozen at -80 °C. Tissue slices of 10 µm were cut using the Cryostar NX70 (Thermo Fisher Scientific, Ghent, Belgium) and placed on glass microscope slides (VWR, PA, USA). For morphometric analyses, optical fields (40 × magnification) of the whole sections were taken by the high content screening microscope Nikon-Marzhauser Slide Express (Märzhäuser Wetzlar GmbH & Co. KG, Wetzlar, Germany) or Vectra Polaris multispectral imaging system (Perkin Elmer, Life Sciences, Zaventem, Belgium) and analysed using QuPath. The images were automatically stitched together to cover the entire slide and were saved as.OME TIFF of.qptiff file format for further analysis.