Multi-marker Immunofluorescence Profiling of Myocardial Samples

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.