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At day 60 (2 months) and 180 (6 months) post-injury, three (3) full-thickness specimens (approximately 3 cm × 1 cm) were harvested from across the scar. Samples were then fixed in 10% neutral-buffered formalin (NBF), embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin (H&E), Masson Trichrome and Verhoeff–van Gieson (VVG) stain. Entire slides were then digitally scanned using the Aperio ScanScope AT2 slide scanner (Aperio Technologies, Vista, CA, USA). Slides were viewed and analyzed using the ImageScope viewer (Aperio Technologies).

Quantitative computer image analysis was performed according the manufacturer’s protocol. All slides were scanned at an absolute magnification of 200× (resolution of 0.25 μm/pixel) using a ScanScope AT2 scanner (Leica Microsystems, Wetzlar, Germany). The obtained digital images of the slides were analyzed using an ImageScope viewer (Version 11.2.0.780; Aperio Technologies, Inc., Vista, CA, USA). CD117⁺ mast cells and CD138⁺ plasma cells were manually counted in 30 random fields for each group (five random fields from each rat) with an area of 0.60 mm² from the digital images of the slides. For the automatic computer analysis of cytokeratin MNF116 and desmin expression, a cytoplasmic v2 algorithm (version 11.2.0.780; Aperio Technologies, Inc.) was used. Using the software, individual stains were calibrated by analyzing sections and recording the average optical density values. The area of analysis was manually determined (Figure 2A,B). The percentage of cells with weak, medium, and strong positive immunostaining were determined for cytokeratin MNF116 (Figure 2C) and desmin (Figure 2D). The total number of cytokeratin MNF116- and desmin-positive cells was counted in 30 random fields for each group (five random fields from each rat), with an average area of 0.28 mm² (for cytokeratin) and 0.12 mm² (for desmin).

ERα- and PR-immunostained slides were scanned at a magnification of 400× (resolution of 0.25 μm/pixel) using the ScanScope AT2 scanner (Leica Microsystems, Wetzlar, Germany). The obtained digital images of the slides were analyzed using the ImageScope viewer (Version 11.2.0.780; Aperio Technologies, Vista, CA, USA). Immunoexpression of ERα and PR in the glandular epithelium and the stroma of the ampulla and isthmus of the fallopian tubes was expressed as a percentage of ERα- and PR-immunopositive cells using automatic computer analysis. For this assessment, a nuclear v9 algorithm (version 9.1; Aperio Technologies, Vista, CA, USA) was applied. Analyzed areas were manually determined. Using the algorithm, the percentage of cells with ERα-positive and PR-positive immunostaining was independently counted in 40 random fields in each group with an average area of 0.4 mm².