Thermo histostar

Explanted samples were washed in PBS and fixed overnight with 4% paraformaldehyde at 4°C. Fixed tissues were processed in a Thermo Excelsior AS (Thermo Fisher Scientific) and embedded with a Thermo HistoStar (Thermo Fisher Scientific) machine. Five-micrometer-thick sections were cut using a Shandon Finesse 325 microtome (Thermo Fisher Scientific). Hematoxylin and eosin, elastic van Gieson, and Masson’s trichrome staining was performed either manually or using a Shandon Varistain 24-4 (Thermo Fisher Scientific) automated machine. Von Kossa staining was carried out using a Silver plating kit (In Vitro Diagnostic Medical Device, Darmstadt, Germany) for the detection of microcalcification. Fibrosis was evaluated by measuring the collagen content of the explants in the Masson’s trichrome staining. Results were expressed as proportion of area occupied by collagen within the graft tissue.

Explanted samples were washed in phosphate-buffered saline and fixed overnight with 4% paraformaldehyde at 4°C. Fixed tissues were processed in a Thermo Excelsior AS (Thermo Fisher Scientific) and embedded with a Thermo HistoStar (Thermo Fisher Scientific) machine. Five-micrometer–thick sections were cut using a Shandon Finesse 325 microtome (Thermo Fisher Scientific). Slides were stored at 37^oC overnight to dry completely before staining. Hematoxylin and eosin and Van Gieson’s stainings were performed using a Shandon Varistain 24-4 (Thermo Fisher Scientific) automated machine. Von Kossa staining was performed using a silver plating kit (In Vitro Diagnostic Medical Device) for the detection of microcalcification. Collagen and elastin contents of the explants in the Van Gieson’s staining were quantified using ImageJ software. Results were expressed as proportion of area occupied by collagen or elastin within the graft tissue.

Explanted samples (Figure 2C) were washed in PBS and fixed overnight with 4% paraformaldehyde at 4°C. Fixed tissues were processed in a Thermo Excelsior AS (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and embedded with a Thermo HistoStar (Thermo Fisher Scientific) machine. Five-micrometer-thick sections were cut using a Shandon Finesse 325 microtome (Thermo Fisher Scientific). Slides were stored at 37°C overnight to dry completely before staining. Hematoxylin and eosin, and Van Gieson’s stainings were performed using a Shandon Varistain 24–4 (Thermo Fisher Scientific) automated machine. Von Kossa staining was carried out using a Silver plating kit (In Vitro Diagnostic Medical Device, Darmstadt, Germany) for the detection of microcalcification. Collagen and elastin contents of the explants in the Van Gieson’s staining were quantified using ImageJ software via color deconvolution, to separate and convert the collagen and elastic fibers to the mean Gray value of the pink and purple and colour intensity, respectively. Threshold values were set for each channel and area-based analysis was used to extract and quantify the regions of interest (ROIs) from the image. Results were expressed as proportion of area occupied by collagen or elastin within the graft tissue.