EMB samples for histological analysis were fixed in 10% buffered formalin and subsequently paraffin-embedded in a tissue processor. 3 μm thick sections were used through the study. The EBM sections were stained with Hematoxylin and Eosin (H&E) according to the standard protocol for the routine histological evaluation. Histological diagnosis was based on the Dallas criteria [30 (link), 31 (link)]. The experienced pathologist evaluated endocardium (thickness, subendocardial fat, fibrosis, and inflammation); myocardium (muscle fiber number, size, and damage); interstitium (fibrosis, fat, edema, and inflammation); and intramural vessels (size, signs of inflammation, damage, and luminal stenosis). Immunohistological assessment of EMBs was carried out as described elsewhere [32 (link)]. Autoantibodies (Santa Cruz Biotechnology, Inc.) against CD3+ (DAKO A0452 Rabbit 1, Hamburg, Germany), CD45Ro (DAKO Hamburg), and CD68+ (DAKO M0876 Mouse 1, Hamburg) were used for immunohistochemical staining. The number of positively stained cells in each biopsy sample was scored by an experienced pathologist and expressed as number of positive cells/mm2. EMB were considered to be inflamed if IHC staining revealed significant inflammatory cellular infiltrates (≥14 leucocytes/mm2 including up to 4 monocytes/mm2 with the presence of CD3 positive T-lymphocytes ≥7 cells/mm2) [33 (link), 34 (link)].
M0876 mouse 1
Manufactured by Agilent Technologies
Sourced in Germany
The M0876 Mouse 1 is a laboratory equipment product manufactured by Agilent Technologies. It is a computer mouse designed for use in a laboratory setting. The product's core function is to provide a pointing and navigation device for interacting with computer systems and software commonly used in laboratory environments.
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2 protocols using m0876 mouse 1
1
Histological Analysis of Endomyocardial Biopsies
2
Immunohistochemical Analysis of Myocardial Inflammation
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Storage of the endomyocardial biopsy samples, and histological and immunohistochemical analyses were performed as described previously [26 (link)]. In brief, we detected antibodies (Santa Cruz Biotechnology, Inc.) against: T-lymphocyte CD3 (DAKO A0452 Rabbit 1, Hamburg, Germany), active-memory T-lymphocyte CD45Ro (DAKO Hamburg), macrophage CD68 (DAKO M0876 Mouse 1, Hamburg), T-helper cell CD4 (DAKO Hamburg, Germany), intracellular adhesion molecule- 1 (ICAM-1) CD54 (NovocastraTM Lyophilized Mouse Monoclonal Antibody CD54 Clone 23G12), and MHC class II cell surface receptor HLA-DR (DAKO Hamburg, Germany). Positive cells were registered by an experienced pathologist and expressed as the number of cells per mm2. Myocardial inflammation was diagnosed according to the criterion established by the ESC Working Group on Myocardial and Pericardial Diseases. This criterion is immunohistochemical detection of significant focal or diffuse cellular infiltration in the endomyocardial biopsy (≥ 14 leucocytes/mm2, including up to 4 monocytes/mm2 with the presence of CD3 positive T-lymphocytes ≥ 7 cells/mm2) [1 (link)]. Inflammatory endothelial activation was diagnosed if immunohistochemical analysis revealed ≥ 3 cells expressing adhesion molecules, i.e., ICAM-1 (CD54) and/or HLA-DR [27 (link)].
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