Vectra 3.0 multispectral imaging system

As described in a previous report [25 ], multiplex immunofluorescence staining was performed using the Opal 7-Color IHC Kit (PerkinElmer, Waltham, MA, USA) in the FFPE tissue sections of a typically paired NSCLC and its brain metastases. The stained slides were scanned by a Vectra 3.0 multispectral imaging system (PerkinElmer, Waltham, MA, USA). The immunofluorescence markers consisted of B7-H4 (1:150 dilution, clone D1M8I, Cell Signaling Technology), CD3 (F7.2.38, prediluted, Dako), CD8 (C8/144B, prediluted, Dako), CD20 (L26, ready-to-use, Leica), CD68 (KP1, prediluted, Dako), and CK (AE1/AE3, prediluted, Dako). DAPI was used for nuclei highlighting. After dewaxing and rehydration, antigen retrievals were performed using a Meidi microwave (Meidi, China). For each primary antibody, tyramide signal amplification linked to specific fluorochrome from the multiplex immunofluorescence staining Kit was used for incubation and visualization. We performed the complete multiplex immunofluorescence staining procedure following the manufacturer’s instructions. In addition, human tonsil FFPE tissues were analyzed with and without primary antibodies following the same multiplex immunofluorescence staining procedure to establish positive and negative (autofluorescence) controls.

Formalin-fixed paraffin-embedded (FFPE) human NP sections as described above were stained with fluorescent multiplex immunohistochemistry for analysis of the expression levels of cGAS and STING^{37 (link),38 (link)}. FFPE NP slides were deparaffinized by xylene, rehydrated by an ethanol gradient, and antigen retrieved by autoclaved Trilogy buffer (CellMarque) for 15 min. The sections were permeabilized by 0.5% Triton X-100 and blocked with 5% BSA. The sections were probed at 4 °C overnight with primary antibody, washed, and incubated for 1 h at 37 °C with the secondary antibody. Then, the sections were labeled with the tyramide-conjugated fluorophore for 10 min. Before the next primary antibody incubation, the sections were heated in 10 mM citric acid for 10 min to wash away the previous antibody. Antibodies paired with Opal tyramide-conjugated fluorophore were used as follows: cGAS and Opal 570, STING and Opal 520. The nuclei were labeled by 0.1 g/ml DAPI. Slices were captured under the Vectra 3.0 multispectral imaging system (PerkinElmer).