Ab 203189

Midpapillary sections of rat hearts were fixed in 4% buffered formalin (ROTI Histofix 4%, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for one week, subsequently embedded in paraffin and cut into slices of 3 μm thickness. The prepared slides were then dewaxed and rehydrated using a standard xylol and a descending alcohol series. Histological examination of the myocardial tissue was performed by means of hematoxylin & eosin staining (Merck KGaA, Darmstadt, Germany) and elastic Van Gieson staining (Merck KGaA, Darmstadt, Germany; Chroma, Waldeck GmbH & Co. KG, Muenster, Germany). All stains were conducted according to a standard protocol.
For immunohistological staining, the ZytoChem-Plus AP Polymer-Kit (Zytomed Systems GmbH, Berlin, Germany) was used according to the manufacturer’s protocol. To detect microthrombi, cluster of differentiation 41 (CD41) antibody (ab 203189, Abcam, Berlin, Germany) and neutrophil myeloperoxidase (MPO) antibody (ab 208670, Abcam, Berlin, Germany) were used as primary antibodies. The evaluation of the stained slides was performed in collaboration with the Department of Pathology, RWTH Aachen University, Faculty of Medicine, Aachen, Germany.

Consecutive staining of 5 markers was performed on 3 µm sections of all myocardial samples on the same slide to enable colocalization assessment as described by Klinge et al. [22 (link)]. Immunofluorescence staining was carried out using primary antibodies for CD41 (ab203189, Abcam, Cambridge, UK), CD68 (ARP63008-P050; Aviva Systems Biology, San Diego, CA, USA), MPO (ab208670, Abcam, Cambridge, UK), and HIF2a (NB100-122, Novus Biologicals, Wiesbaden, Germany) according to a standard protocol. A secondary antibody, ImmPRESS^® HRP Horse Anti-Rabbit IgG Polymer Detection Kit-Peroxidase (MP-7401; Vector Laboratories, Newark, NJ, USA), was used and coupled with an Opal dye, respectively. CD41 was labeled with Opal 480 (P1500001KT, Akoya Biosciences, Menlo Park, CA, USA), HIF2a with Opal 520 (FP1487001KT, Akoya Biosciences, Menlo Park, CA, USA), CD68 with Opal 570 (FP1488001KT, Akoya Biosciences, Menlo Park, CA, USA), and MPO with Opal 650 (FP1496001KT, Akoya Biosciences, Menlo Park, CA, USA). Hypoxyprobe™ was included as the fifth marker in this panel, as described below.