Cd15 carb 3

All specimens were fixed in 10% neutral formalin, entrapped through conventional paraffin embedding, and processed into 4 μm serial sections with conventional HE staining. The immunohistochemical S-P method was employed to mark immune phenotypes. LCA (2B11, DAKO), CD3 (F7.2.38, DAKO), CD45RO (OPD4, DAKO), CD20 (L26, DAKO), CD10 (MX002, DAKO), CD15 (Carb-3, DAKO), CD30 (Ber-2, DAKO), Bcl-2 (SP66, DAKO), Bcl-6 (LN22, AKO), CyclinD-1 (DCS-6, DAKO), PAX-5 (SP34, DAKO), kappa light chain (L1C1, DAKO) and lambda light chain (LAM03+HP6054, DAKO) were used.

Tissue sections (4 µm thick) from archived paraffin-embedded tissue blocks were prepared for immunohistochemical and hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed using antibodies against CD3 (2GV6; Roche, Basel, Switzerland), CD20 (L26; DAKO, Santa Clara, CA), CD15 (Carb-3; DAKO), CD123 (6H6; Thermo Fischer Scientific), CD163 (10D6; Leica Microsystems, Wetzlar, Germany), or MPO (polyclonal; DAKO). All staining procedures were performed using an autoimmunostainer (Bond III [Leica Microsystems] or BenchMark Ultra [Ventana Medical Systems, Oro Valley, AZ]).

We performed tyramide signal amplification labeling with the Opal reagents (PerkinElmer, Waltham, Massachusetts) using the Opal 4‐color automation IHC method. The primary antibodies used were as follows; LOX‐1 (ab126538, 1:800, Abcam, Cambridge, UK), CD15 (Carb‐3, 1:400, Dako, Glostrup, Denmark), CD11b (ab52478, 1:400, Abcam, Cambridge, UK), CD45 (1:400, #13917, Cell Signaling Technology, Inc., Danvers, Massachusetts), CD3 (ab16669, 1:300, Abcam, Cambridge, UK), CD20 (L26, 1:400, Dako, Glostrup, Denmark), CD163 (1:1000, #93498, Cell Signaling Technology, Inc., Danvers, Massachusetts), and α‐SMA (M0851, 1:100, Dako, Glostrup, Denmark). Antigens were retrieved from the tissue sections using ImmunoSaver (Nisshin EM, Tokyo, Japan). Tissue sections were incubated with fluorophores Opal 520, 570, and 690 for 10 minutes at room temperature. Antigen retrieval was performed using 10 mM sodium citrate buffer pH 6 in a microwave for 15 minutes. Finally, all sections were counterstained with DAPI. Images were captured using the BZ‐X700 microscope (Keyence). We analyzed 10 cases from the stromal LOX‐1 high group using triple‐labeled high‐power fields.