Cd5 clone 4c7

Skin biopsy specimens were routinely fixed in a 10% neutral-buffered formalin solution, and embedded in paraffin. The 4.5-µm sections of FFPE blocks were stained by H&E. Immunohistochemical analysis using the biotin-free alkaline phosphatase method (Leica Biosystems, Newcastle, UK) was performed using the fully automated stainer BOND-III systems (Leica Biosystems) with the following primary antibodies: CD3 (clone LN10, Leica Biosystems), CD4 (clone 4B12, DakoCytomation, Glostrup, Denmark), CD5 (clone 4C7, Leica Biosystems), CD20 (clone LR26, Leica Biosystems), CD56 (clone NCAM, Leica Biosystems), CD68 (clone 514H12, Leica Biosystems), CD123 (clone BR4MS, Novocastra, Leica Biosystems), TCL-1 (clone MRQ-7, Cell Marque, CA Rocklin, USA), TIA-1 (Biocare Medical, CA Pacheo, USA), and MPO (Cell Marque). Irrelevant IgG subclass‒matched antibodies were used for negative controls.

CD8, FOXP3, PD‐1, CD117 and CD5 immunohistochemistry was performed in the eight MNT and three MNCA cases. PD‐L1 immunohistochemistry was performed in all the 35 thymic epithelial neoplasm cases. Antigen retrieval for CD8, FOXP3 and PD‐L1 was performed with the buffer preheated to 95°C for 40 min (pH 9.0). Antigen retrieval for PD‐1, CD117 and CD5 was conducted with preheating to 120°C for 15 min (pH 6.0). The antibodies against these antigens were as follows: CD8 (clone 4B11, 1:40; Leica Biosystems, Wetzlar, Germany), FOXP3 (clone 236 A/E7, 1:100; Abcam, Cambridge, UK), PD‐1 (clone NAT105, 1:50; Abcam), PD‐L1 (clone E1L3N, 1:100; Cell Signaling Technology, Danvers, MA, USA), CD117 (polyclonal, 1:500; DakoCytomation, Carpinteria, CA, USA) and CD5 (clone 4C7, 1:400; Leica Biosystems). All sections were incubated with primary antibodies for 30 min. They were then incubated with peroxidase‐labeled goat anti‐mouse/rabbit IgG antibody (Nichirei Biosciences, Tokyo, Japan) for 30 min. The immune response was visualized using 3,3′‐diaminobenzidine (DakoCytomation) and counterstained with hematoxylin. Two board‐certified pathologists (HY and TI) examined the hematoxylin and eosin and immunohistochemical stains for CD5 and CD117.