Immunofluorescence Staining of MSC Aggregates

MSC aggregates were cultivated in Nunc^TM Lab-Tek^TM II chamber slides (Nunc, Thermo Fisher Scientific, USA), fixed and permeabilized after 16 hours and stained with Alexa Fluor AF^® 594 phalloidin 1:200 (0.1 U/ml, Molecular Probes, Thermo Fisher Scientific, USA) to reveal filamentous (f)-actin. To stain focal adhesions, a mouse mAb to paxillin and a mouse mAb vinculin both 1:100 (both from Santa Cruz Biotechnology, USA) followed by goat-anti-mouse Fab fragments labeled with AF^® 488 1:500 (3 μg/ml, Jackson Laboratories, USA) were used, and nuclei were stained with DAPI 1:1000 (Sigma-Aldrich, USA). As endothelial marker von Willebrand factor (vWF) mAb (2 μg/ml, Dianova, Germany) 1:100, followed by goat-anti-rabbit Fab fragments labeled with AF^® 488 1:500 (3 μg/ml, Jackson Laboratories, USA) were used. The slides were mounted with Fluoromount-G^TM (Southern Biotechnology, Thermo Fisher Scientific, USA) and analyzed with an Apochromat 63x objective on a confocal microscope (TCS SP8, Leica Microsystem GmbH, Germany) using the LASX-software version 3.1.5–16308 [21 (link)].