Mca1211

Myocardial tissue was embedded with O.C.T., cut into 10-μm-thick sections, and fixed with acetone. For staining of platelets, a rat anti-mouse CD41 antibody was used (GTX 76011, GeneTex, Irvine, CA), and for control purposes, an IgG1 isotype control was used (MCA1211, Serotec, Puchheim, Germany). Secondary staining was performed with a biotinylated rabbit anti-rat IgG (BA-4001, Vector). Alkaline phosphatase and substrate kit (AK-5000 & SK-5100, Vector) and levamisole (X3021, DAKO) were used for detection. Finally, samples were embedded with Kaiser’s glycerol gelatin (Merck, Darmstadt, Germany) until adequate staining.
Platelets were counted in two representative pictures of × 20 magnification out of the inflamed myocardium of two different CD41–stained slices. For counting MPIOs, the inflamed myocardium of 10 representative CD41 stained slices was examined carefully.

Platelets were stained using a rat anti-mouse CD41 antibody (GTX 76011, GeneTex, Irvine/CA, USA) or unspecific control IgG1 isotype (MCA1211, Serotec, Puchheim, Germany). For secondary staining a biotinylated rabbit anti-rat IgG (BA-4001, Vector, Burlingame/CA, USA) was used. Afterwards, alkaline phosphatase and substrate kit (AK-5000 & SK-5100; Vector, Burlingame/CA, USA) after levamisol pre-treatment (X3021, DAKO, Hamburg, Germany) was applied for antibody detection. Sections were then embedded in Kaiser’s Glyceringelatine (1092420100, Merck, Hamburg, Germany). Due to their size (1 μm), MPIO beads are visible using 63x magnification without further staining. MPIOs were quantified as mean number of MPIOs of 3 sections per section at 63x magnification.
Histology sections were analyzed using a light microscope (Zeiss optics, Germany). Pulmonary embolism was quantified as mean of 3 representative sections of the according. Pulmonary emboli were graded according to size of obstructed vessels: small vessel (maximum diameter < 150 μm: GI < 50% vessel obstruction, GII > 50% vessel obstruction) or large vessels (maximum diameter > 150 μm: GIII < 50% vessel obstruction, GIV > 50% vessel obstruction).