Cd8 clone c8 144b

Immunohistochemical (IHC) procedures were conducted as described previously [15 (link)]. Antibodies used are PD-L1 (SP142, rabbit monoclonal, Spring Bioscience, Pleasanton, CA, USA), PD-1 (NAT105, mouse monoclonal, GeneTex, Irvine, CA, USA), CD4 (clone SP35, rabbit monoclonal, Spring Bioscience, Pleasanton, CA, USA), CD8 (clone C8/144B, mouse monoclonal, Nichirei Biosciences, Tokyo, Japan), CD68 (PG-M1, mouse monoclonal, DAKO, Tokyo, Japan), and VEGF (A-20, rabbit polyclonal, Santa Cruz, Dallas, TX, USA). For PD-L1 staining, antigen retrieval was done by autoclaving at 121 °C for 10 min in Tris/EDTA buffer (pH 9.0), and 1st antibody incubation (1:100) was conducted at 4 °C overnight. The corresponding normal endometria or stroma provided an internal positive control, and negative controls without addition of primary antibody showed low background staining.

Immunohistochemical staining was performed on 3 μm-thick sections obtained from paraffin-embedded tissues. Mouse monoclonal antibodies against CD68 (clone PG-M1; Dako, Glostrup, Denmark), CD8 (clone C8/144B; Nichirei, Tokyo, Japan) and CD169 (clone HSn7D2; Santa Cruz Biotechnology, CA) were used. After the samples were reacted with primary antibodies, they were incubated with horseradish peroxidase-labeled secondary anti-mouse antibody (Nichirei, Tokyo, Japan). Signals were visualized using the diaminobenzidine system (Nichirei, Tokyo, Japan). Normal mouse immunoglobulin (DAKO, Glostrup, Denmark) was used as a negative control and no signal was observed in those control sections. Data regarding the Ki-67 labeling index, as well as the statuses of ER, PgR, and Her2, were previously evaluated by our research group [19 (link), 20 (link)]. CD68-, CD169-, and CD8-positive staining was counted in four randomly selected fields-of-view by two pathologists (TS and YM) who were blind to patient information. The average numbers of counted cells were determined and were used to calculate the number per mm². To evaluate lymph node metastasis, sinus macrophages were counted in non-metastatic regions. The detailed methods of cell counting in RLNs have been previously described [21 (link)].