M361929 2

FISH was performed on CSCC tissue microarrays using a fluorescence ISH kit (Boster BioTech) and the miR-142-5p synthetic oligonucleotide probes (Exiqon), according to the manufacturer’s protocol. In brief, the slides were incubated with 3% hydrogen peroxide (H₂O₂), digested with pepsin for 2 min at 37 °C, and fixed with 1% paraformaldehyde (PFA) in diethyl pyrocarbonate (DEPC) for 5 min. The slides were then pre-hybridised in a hybridisation buffer at 42 °C with miR-142-5p or U6 probes and incubated with fluorescent streptavidin-biotin complex (f-SABC) and horseradish peroxidase polymer. For IHC, sections were immersed in 3% H₂O₂ to block endogenous peroxidase activity and incubated with primary antibodies, overnight at 4 °C. A horseradish peroxidase-conjugated anti-rabbit secondary antibody (ZSGB BioTech) was subsequently applied for 1 h at room temperature. The expression of D240 (M361929-2, DAKO), IDO (86630, CST), and CD8 (ab93278, Abcam) was visualised using 3,3′-diaminobenzidine (DAB) following counterstaining with haematoxylin (ZSGB BioTech). FISH- and IHC-stained tissue sections were reviewed and scored separately by two independent pathologists. For semi-quantitative evaluation of IDO and miR-142-5p expression in tissue sections, the German Immuno-Reactive Score was applied as previously described [23 (link)].

An Opal 7-color Fluorescence Immunohistochemistry (IHC) Kit (PerkinElmer, Waltham, MA) was utilized for the mIHC assay, with the following primary antibodies involved: anti-CD8 (1:800, ab93278, Abcam), PD-1 (1:800, ab137132, Abcam), D240 (1:500, M361929-2, Dako, Glostrup, Denmark) and PD-L1 (5 μg/mL, ab205921, Abcam). Sections were incubated with the HRP Broad Spectrum SuperPicture Polymer Detection Kit (Thermo Fisher Scientific) and diluted with opal fluorescent dyes (Opal520, Opal570, Opal 620 and Opal 690). Afterwards, the sections were microwaved with AR6 buffer, and incubated with DAPI, followed by observation under a Zeiss LSM 880 confocal laser scanning microscopy.