Clone pg m1

Endomyocardial biopsies were performed in all patients according to current ESC diagnostic guidelines [4 (link)]. At least five biopsies were taken, fixed in 4% phosphate-buffered formaldehyde, and embedded in paraffin. Four µm thick tissue samples were stained with Masson’s trichrome, hematoxylin-eosin as well as Giemsa and examined by light microscopy.
For immunohistological detection of cardiac immune cells, a monoclonal rabbit-anti-CD3 antibody (Clone SP7, 1:500, Novocastra Laboratories, Newcastle upon Tyne, United Kingdom), a monoclonal mouse anti-human CD68 antibody (Clone PG-M1, 1:50) and a monoclonal mouse anti-human HLA-DR alpha-chain antibody (clone TAL.1B5, 1:50), both from DAKO, Hamburg, Germany, were used. Immunohistochemical analysis was performed on an automated immunostainer following the manufacturer’s protocol (Benchmark; Ventana Medical Systems, Tucson, AZ, USA) and using the ultraView detection system (Ventana) and diaminobenzidine as substrate. Tissue sections were counterstained with hematoxylin. The detection of >14 infiltrating leukocytes/mm² (including >7 CD3+ T-lymphocytes and/or CD68+ macrophages) in the presence of myocyte damage and/or fibrosis in addition to enhanced human leukocyte antigen class II expression in professional antigen-presenting immune cells and endothelium was used for the diagnosis of myocarditis [9 (link)].

Formalin-fixated paraffin-embedded blocks of renal tumors were analyzed by immunohistochemistry. Heat-induced epitope retrieval was performed with EDTA for CD3 and with Trilogy for PD-L1, PD-1 and CD68. Staining was performed on a DAKO autostainer plus. After blocking endogenous peroxidase, sections were incubated for 45 min with rabbit monoclonal anti-PD-L1 antibody (1:1000; Cell Signaling #13,684, clone E1L3N), mouse monoclonal anti-CD68 antibody (1:100; Dako #M0876, clone PG-M1), mouse monoclonal anti-CD3 antibody (1:50; Dako #M7254, clone F7.2.38) and rabbit polyclonal anti-PD-1 antibody (1:150; Zytomed #516-18,662). Sections were washed and incubated with Dako REAL EnVision HRP Rabbit/Mouse polymer, which reacts with DAB-Chromogen, according to the manufacturer’s protocol. Different pathologic scores were determined [24 (link)–26 (link)], including tumor cell staining score (TPS-score), immune cell staining (IC-score) and a combined score (CPS-score) for PD-L1 and the immune cell staining scores (IC-score) for PD-1, CD3 and CD68, respectively. Total RNA was extracted from slices cut in line from the immunohistochemistry blocks (High Pure FFPE RNA Micro Kit, Roche Diagnostics GmbH, Germany).

All plaques specimens were examined by immunohistochemistry for the expression of ST2L receptor in the whole section using a rabbit polyclonal anti-ST2 antibody (IL1RL1 interleukin receptor 1), (1:500 dilution; Sigma Aldrich). All immunostainings were done by an automated immunostainer (Dako Autostainer). Slices were pretreated using the DAKO-PT-link in a pH 9 EDTA retrieval solution. Substitution of the primary antibody with PBS served as a negative control. Colonic biopsies of patients affected by IBD were used as a positive control. Immunostainings for CD68 (clone PGM-1, 1:100 dilution, DAKO-Denmark), CD 45 (LCA, 1:500 dilution, DAKO-Denmark) and Tryptase (clone AA1, 1:150 dilution, DAKO) were also performed to evaluate the presence of inflammatory cells in the plaque, namely macrophages (CD 68 positive cells), lymphocytes (CD 45 positive cells) and mastocytes (tryptase positive cells).

For detection of adipocytes and macrophages simultaneously, tissue sections were heat pretreated with citrate buffer and incubated with perilipin1 (PLIN1 (D1D8) XP® Rabbit monoclonal antibodies (mAb), Cell Signaling Technology, Germany) and CD68 (anti-human-CD68 mAb, Clone PG-M1, Dako, Denmark) antibodies. Primary antibodies were visualized with the secondary antibodies donkey-anti-rabbit-Alexa-Fluor-Plus 555 (Invitrogen, California) and donkey-anti-mouse-Alexa-Fluor-Plus 488 (Invitrogen, California). Image acquisition was conducted with Celena S Digital imaging System (Logos Biosystems, South Korea).

For immunohistochemistry, 4μ-thick sections obtained from formalin-fixed/paraffin-embedded tissues were placed on positive-charged electrostatic slides (Isotherm Technical Laboratory Glass Materials). All sections were then placed on a fully automated immunohistochemical staining machine (Ventana, Benchmark, XT IHC/ISH). UltraView Universal DAB Detection Kit compatible with the device was used for IHC staining. Monoclonal mouse primary antibodies CD163 (clone MRQ-26, prediluted, VENTANA, Cat. No: 760-4437) and CD68 (clone KP-1, prediluted, VENTANA, Cat. No: 790-2931and clone PG-M1, prediluted, DAKO, Code: IS613, prediluted, Cat. No: 760-4437) were applied and staining was completed according to the standard procedures. To detect EBV-RNA with an EBER (Epstein-Barr virus-encoded RNA) probe via the CISH method, 4μ-thick tissue sections placed on positive-charged electrostatic slides were also used. All tests were performed with a fully automated ISH machine (Ventana, Benchmark XT, IHC/ISH), by using EBER 1 DNP Probe (Regulatory status: ASR; Cat. No: 760-1209), compatible with the machine, and the Ultraview AP Red ISH Kit for EBV RNA signaling, and staining was completed according to the standard procedures.

Tissue sections were cut, placed onto glass slides, de-waxed in xylene and alcohol and then washed with phosphate-buffered saline twice as our previous study. Tissue sections were stained with mouse anti-human CD68 monoclonal antibody (Clone PG-M1 from Dako, Glostrup, Denmark) or rabbit anti-human S100A9 monoclonal antibody (GeneTex) separately. The five most representative high-power fields (200 magnified) per slide were manually selected using an Olympus BX50 microscope (Olympus, Southall, United Kingdom). The nuclear cells with positive CD68 or S100A9 staining were counted by ImageJ (NIH) by independent pathologist blinded to clinical outcome. Median number of positive CD68 or S100A9 cells were use to divide the high and low expression group.