Cd4 clone sp35

The data of CD4+, CD8+, and FOXP3+ TILs and PD-L1+ ICs were adopted from our previous studies [25 (link), 26 (link)] for all of the cases of pre-invasive carcinoma and 307 cases of invasive carcinoma. Immunohistochemical staining had been carried out using the following antibodies: CD4 (clone SP35; ready to use; Dako), CD8 (clone C8/144B; ready to use; Dako), FOXP3 (clone 236A/E7; 1:100; Abcam) and PD-L1 (clone E1L3N; 1:100; Cell Signaling, Danvers, MA, USA). CD4+, CD8+, and FOXP3+ T cells had been counted in intratumoral and stromal areas as absolute numbers per high-power field. Detailed information on the counting method of TILs is described in the previous studies [25 (link), 26 (link)]. For this study, CD4+, CD8+, and FOXP3+ TILs were dichotomized into high- and low-infiltration groups using cutoff values obtained by ROC curve analyses. PD-L1+ ICs were considered to be present when at least 1% of the tumor stromal area was occupied by PD-L1+ ICs.

The data for the CD4+, CD8+, and FOXP3+ TILs and PD-L1+ immune cells were adopted from our previous studies^{11 (link),36 (link)} for 288 cases of DCIS and 339 cases of IBC. Immunohistochemical staining had been performed with a BenchMark XT autostainer (Ventana Medical Systems) using an UltraView detection kit (Ventana Medical Systems). The following antibodies were used: CD4 (clone SP35; ready to use; Dako), CD8 (clone C8/144B; ready to use; Dako), FOXP3 (clone 236A/E7; 1:100; Abcam) and PD-L1 (clone E1L3N; 1:100; Cell Signaling, Danvers, MA, USA).
CD4+, CD8+, and FOXP3+ T cells had been counted in intratumoral and stromal areas as absolute numbers per high-power field. Detailed information on the counting method of TILs is described in the previous studies^{11 (link),36 (link)}. PD-L1+ immune cells were considered to be present when at least 1% of the tumor stromal area was occupied by PD-L1+ immune cells.

Formalin-fixed paraffin-embedded tissues were stained with hematoxylin and eosin at the initial diagnosis. Immunohistochemistry was performed using the following panel of monoclonal and polyclonal antibodies: CD20 (clone L26, DAKO, Glostrup, Denmark); CD79a (clone 1.10E + 04, Leica Biosystems, Wetzlar, Germany); PAX5 (clone R1, DAKO); CD10 (clone 56C6, Leica Biosystems); BCL6 (clone P1F6, DAKO); MUM1 (clone MUM1p, DAKO); Ki-67 (clone MIB-1, DAKO); Terminal deoxynucleotidyl transferase (TDT, clone SP150, DAKO); CD2 (clone AB75, DAKO); CD3 (clone LN10, Leica Biosystems); CD4 (clone SP35, DAKO); CD7 (DAKO); CD8 (clone SP16, DAKO); CD1a (clone SP157, DAKO); CD34 (clone QBEnd/10, DAKO); CD43 (clone DF-T1, DAKO); CD99 (clone HO36-1.1, DAKO); CD117 (clone C-KIT, DAKO); CD45 (clone PAN-LCAL, DAKO); CD33 (clone WM-54, DAKO); MPO (clone SP72, DAKO); CD15 (clone C3D-1, DAKO); CD30 (clone Ber-H2, DAKO); CD23 (clone SP23, DAKO); and Epithelial membrane antigen (EMA, clone GP1.4, Leica Biosystems).