Picrosirius red

The apex of the harvested hearts was embedded in gum tragacanth (Sigma-Aldrich Chemie GmbH, Germany) and then stepwise frozen in isopentane and liquid nitrogen. Embedded tissue was stored at −80 °C until further usage. A cryotome (Leica CM 3050 S, Leica Microsystems GmbH, Wetzlar, Germany) was used for sections that were stained with haematoxylin and eosin, and Picrosirius Red as previously described [6 (link),8 (link),26 (link),59 (link)]. For Picrosirius Red stain, 6µm thick myocardial sections were fixed in ice-cold acetone for 10 min and afterwards washed twice with 98% ethanol for 5 min. Slides were then stained for 30 min in Picrosirius Red F3BA (Polysciences, Inc., Warrington, PA, USA). After rinsing with distilled water, slides were washed with 98% and absolute ethanol, dehydrated with xylol and mounted with Vitro-Clud (R. Langenbrinck GmbH, Emmendingen, Germany). Images were acquired using a Leica CTR 6500 HS microscope (Leica Microsystems GmbH, Wetzlar, Germany). Fibrosis was quantified using ImageJ software 1.42c (http://rsb.info.nih.gov/ij, last access 29 April 2022). In tile scan images, the borders of the myocardium were selected manually, and staining artefacts were removed. Using a predefined threshold, the red area representing fibrosis was measured and the results are presented as a percent of the total area.

For the differentiation screening, hydrogels were first washed with two consecutive 10 min washes of fresh PBS. Following the PBS washes, the cell-laden constructs were placed in cassette sample holders and submerged in optimal cutting temperature (OCT) solution for 30 min. Subsequently, samples were snap frozen in liquid nitrogen, and 10-µm slices were sectioned on a cryostat machine and stained for safranin-O and picrosirius red to analyze the GAG production and collagen production, respectively. Briefly, for safranin-O staining, frozen sections were first let to air dry, rehydrated, and then immersed in 0.25% safranin-O solution for 7 min. Sections were then rinsed with water, fixed in methanol, dehydrated via graded ethanol, and mounted. For picrosirius red staining, air dried sections were rehydrated and placed in a picrosirius red solution (Sigma) for 1 h at room temperature. Next, stained sections were washed twice in fresh acidified water, fixed in methanol, dehydrated in graded ethanol, and finally, mounted. Brightfield images of safranin-O and polarized images of picrosirius red stained sections were taken using a DMR microscope (Leica Microsystems, Diegem, Belgium). For all sections, images were taken using a 10× objective.