Cd20 l26

Immunohistochemical detection of survivin was performed using a Dako Omnis autostainer (Dako North America, Inc. Carpinteria, CA) with rabbit monoclonal survivin antibody clone EP119 (Bio SB, Santa Barbara, CA). Additional antibodies included: CD4 (4B12, Dako); CD8 (C8/144B, Dako); CD20 (L26, Dako); PD-L1/CD274 (SP142, Spring Bioscience). Stained specimens were viewed by the neuropathologist, co-investigator (JQ), and survivin expression in the nucleus and cytoplasm was determined to be present or absent.

Tissue microarrays containing the specimens from 30 CRC patients who received curative surgery in the first affiliated hospital of Chongqing medical university (Chongqing, China) from April 2016 to September 2017 were constructed for immunohistochemistry. Specimens were all confirmed by pathological analysis as colorectal cancer. Immunohistochemistry was performed as described earlier,32, 33 using monoclonal antibodies against CD3 (SP7), CD8 (4B11), CD45RO (OPD4), CD57 (NK1), tryptase (AA1), CD1A (Ab‐5), granulocytes (BM‐2), PDPN (D2‐40), cytokeratin (AE1AE3), FOXP3 (ab20034; AbCam, Cambridge, United Kingdom) CD68 (PGM‐1), CD20 (L26; DAKO, Carpinteria, CA), IL3RA (IL3RA; ATLAS Antibodies, Stockholm, Sweden), CXCR5 and IL‐17 (H‐132; Santa Cruz Biotechnology, Santa Cruz, CA). Isotype‐matched mouse monoclonal antibodies were used as negative controls. Slides were analyzed using an image analysis workstation (Spot Browser, ALPHELYS). Polychromatic high‐resolution spot‐images (740 × 540 pixel, 1.181 μm/pixel resolution) were obtained (x200 fold magnification). The density was recorded as the number of positive cells per unit tissue surface area. For each duplicate, the mean density was used for statistical analysis.

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.

Immunohistochemistry was performed on 2‐μm FFPE sections in an automated immunostainer (Autostainer Link48; Dako, Glostrup, Denmark) according to the manufacturer's instructions. The following antibodies were used: β‐catenin 14 (1 : 1000; BD Transduction, South San Francisco, CA, USA), CD3 polyclonal (1 : 400; Dako), CD4 4B12 (1 : 300; Dako), CD8 C8/144B (1 : 20; Dako), CD20 L26 (1 : 400; Dako), CD21 1F8 (1 : 50; Dako), CD56 123C3 (1 : 400; Dako), CD68 KP1 (1 : 3000; Dako), CD163 10D6 (1 : 200; Novocastra, Newcastle, UK), FOXP3 259D/C7 (1 : 200; BD Pharmingen), PD‐L1 SP142 (1 : 30; SPRING, Pleasanton, CA, USA), PD1 NAT105 (1 : 100; Biocare Medical, Pikenine, Concord, NC, USA), and EZH2 5246S (1 : 50; Cell Signaling, Leiden, Netherlands).

Staining was carried out on FFPE sections following deparaffinization and antigen retrieval. Endogenous peroxidase was blocked with 3% hydrogen peroxide. The sections were incubated with protein block (DAKO) and stained with primary antibodies against CD8 (SP16, Spring Bioscience), CD20 (L26, Dako) and DC-LAMP (1010E1.01, Dendritics), followed by the manifestation of enzymatic activity and hematoxylin counterstaining. The images were acquired using a Leica Aperio AT2 scanner (Leica).