Opal multiplex ihc assay kit

Opal Multiplex IHC assay kit (PerkinElmer, USA) was used for mIHC staining according to the literature [30 (link)]. Slides were scanned with a Pannoramic MIDI slide scanner (3DHISTECH, Hungary) at 20 × magnification and analyzed with HALO image analysis platform (Indica, USA). The relationship of CD8⁺ T cells with tumor vasculature was analyzed as reported [31 (link)].

For polychromatic immunofluorescence, the Opal Multiplex IHC Assay Kit (PerkinElmer, USA) was employed on formalin-fixed paraffin-embedded FIA sections following the manufacturer's instructions. Fluorescent images were acquired using a Zeiss Axio Scope A1 microscope. Primary antibodies included: anti-MMP-9 (1:20, BM4089, Boster), anti-α-SMA (1:20, BM3902, Boster), anti-ICAM1(1:50, A00171, Boster), anti-p-JAK2 (1:500, bs-3206R, Bioss), anti-p-Src (1:50, bs-1730R, Bioss), anti-p-PLCγ (1:100, #MAB74542, R&D systems), anti-p-Erk1/2 (1:100, bs-3016R, Bioss), anti-p-STAT1 (1:50, #7649, CST), anti-p-STAT3 (1:20, BM4835, Boster). Rabbit IgG (#3900, CST) or mouse IgG1 (#5415, CST) with the same concentration as primary antibodies were utilized as isotype controls to validate antibody specificity. Meanwhile, secondary antibody only controls were used to distinguish genuine target staining from background.