Stainer

The sections of TMA were stained with VISTA, CD3, CD4, CD8, and CD19 antibodies. The rabbit IgG monoclonal antibody (D1L2G; dilution1:200, Cell Signaling Technology, USA) was used to detect the expression of VISTA. Human placental and tonsil tissues obtained from our hospital were used as positive control samples. SignalStain^®Antibody Diluent #8112 and SignalStain^®Boost (HRP, Rabbit) #8114 were used as diluent and detection reagents, respectively. The details of primary antibodies and immunohistochemistry were described in Supplementary Table 1. All sections were stained using the Leica stainer (Leica Biosystems, Germany) complying with the instructions. Two pathologists (XYR and HWW) independently reviewed the immunohistochemical staining and scored for each sample. The consensus of the two observers was more than 90%. Less than 10% of the sections had inconsistent results which were resolved via the joint evaluation of the particular tumor area.

Formalin-fixed paraffin-embedded (FFPE) samples were collected for each patient, with FFPE tissue sections prepared at a thickness of 4 μm. Seven slides of serial sections were collected per sample and stained for CD3, CD4, CD8, CD19, CD163, and cytokeratin (AE1/AE3), as well as H&E staining in order. IHC was performed according to the manufacturer’s protocol using a Leica stainer (Leica Biosystems, Germany). ER, PR, Her-2, P53, Ki67 were stained at time of diagnosis as previously described (14 (link)). HER-2 (2+) cases were further subjected to fluorescent in situ hybridization (FISH) to confirm its negative. Details of the primary antibodies of CD3, CD4, CD8, CD19, CD163, AE1/AE3 and their respective optimizations are listed in Supplementary Table 1. After staining, all the slides were scanned with a KF-pro-400 (Ningbo, China) scanner under the same acquisition conditions, with a magnification of 40× (0.2 μm/pixel). The images were then transformed into digital image files.