Pd 1 clone nat105

Formalin-fixed biopsies were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate pH 6.0 (for HbcAg), Tris-EDTA pH 9.0 (for CD4 and CD8), pepsin (for CD68), or commercial CC1 buffer Ventana (for PD-1 and PD-L1). Sections were then incubated with antibodies to the following: HBsAg (clone A10F1, 1:100, Cell Marque, Rocklin, CA), HbcAg (1:800, Abcam, Cambridge, UK), CD4 (clone 4B12, 1:200, Leica, Buffalo Grove, IL), CD8 (clone 4B11, 1:1000, Leica, Buffalo Grove, IL), CD68 (clone PG-M1, 1:300, Dako, Santa Clara, CA), PD-1 (clone NAT105, 1:200, Abcam, Cambridge, UK), PD-L1 (clone 28-8, 1:200, Abcam, Cambridge, UK). Expression of the markers was quantified using the Visiopharm software, by dividing the number of strongly positive cells by the total stained area. PD-L1 staining on hepatocytes was scored on a semi-quantitative scale: 1+ 25% positive, 2+ as intermediate, and 3+ diffuse staining.

Formalin‐fixed and paraffin‐embedded (FFPE) tissue samples of human colon cancer moderately differentiated (G2) cases were selected for in situ immunophenotypic analyses. Four‐micrometers‐thick sections were deparaffinized, rehydrated, and unmasked using Novocastra Epitope Retrieval Solutions pH 9 in a thermostatic bath at 98 °C for 30 min. Subsequently, the sections were brought to room temperature and washed in PBS. After neutralization of the endogenous peroxidase with 3% H₂O₂ and Fc blocking by a specific protein block (Leica Novocastra, Wetzlar, Germany), the samples were incubated with phospho‐DRP1 (Ser 616) (clone D9A1 Cell Signaling, 1 : 100) and PD‐1 (clone NAT105 Abcam, 1 : 50) antibodies. IHC staining was revealed using MACH 2 Double Stain 1 kit (Biocare, Pacheco, CA, USA) and DAB (3,3'‐diaminobenzidine, Leica Novocastra) and Vulcan Fast Red as substrate chromogens. Triple immunostainings were performed by incubating the same sections with CD8 antibody (clone 4B11, 1 : 50 pH9, Leica Novocastra) and anti‐mouse Alexa Fluor 488‐conjugated secondary antibody (1 : 500, Life Technologies, Carlsbad, CA, USA). Slides were analyzed under a Zeiss Axioscope A1, and microphotographs were collected using a Zeiss Axiocam 503 Color with the Zen 2.0 Software (Zeiss).

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.