Hpa002822

Multicolor immunofluorescence staining was performed in tissue sections of BC surgically resected from five patients to detect EMILIN1, CD8, and TGFBI expression and from seven patients to detect EMILIN1, CD8, and Ki-67 using the Akoya Biosciences Opal Kit following the manufacturer's instructions. All patients were selected from the validation group of BC samples. In the first five samples, EMILIN1 staining (anti-EMILIN1 rabbit polyclonal antibody: x400, HPA002822, Sigma) was visualized using the Opal 480 Fluorophore; CD8 staining (anti-CD8 rabbit polyclonal antibody: x500, ab4055, Abcam) with the Opal 570 Fluorophore; and TGFBI staining (anti-TGFBI rabbit polyclonal antibody: x400, 10188-1-AP; Proteintech, Rosemont, IL, USA) with the Opal 520 Fluorophore. In the other seven BC samples, EMILIN1 staining was visualized using the Opal 480 Fluorophore, CD8 staining with the Opal 570 Fluorophore, as above, and Ki-67 staining (anti-Ki67 rabbit monoclonal antibody: x500, #9027; Cell Signaling Technology, Danvers, MA, USA) with the Opal 520 Fluorophore. All sections were lightly counterstained with hematoxylin and examined under an All-in-One BZ-X710 fluorescence microscope (KEYENCE Corporation, Osaka, Japan).