Trichrome 3 blue staining kit

In order to evaluate the fibrotic surface area, Masson’s trichrome staining was performed using the Roche Trichrome III Blue Staining Kit (Roche, Basel, Switzerland). The fibrotic surface, nuclei, and cytoplasm were stained blue, black, and red, respectively. The slides were visualized by light microscopy at 100× magnification, and fibrosis relative surface area were analyzed using i-Solution image analysis software (IMT i-Solution Inc., Daejeon, Korea).

Masson’s trichrome staining was performed to measure fibrosis in the OT graft using the Roche Trichrome III Blue Staining Kit (Roche, Basel, Switzerland). The fibrotic surface, nuclei, and cytoplasm space were stained as blue, black, and red, respectively. The stained slides were scanned with a Leica slide scanner at × 200 magnification, and analyzed using the i-Solution image analysis software for fibrotic surface area analysis.

Masson’s trichrome staining was performed to analyze ovarian stromal fibrosis using the Roche Trichrome III Blue Staining Kit (Roche, Basel, Switzerland). After deparaffinization and rehydration, the slides were treated with Bouin solution (Sigma-Aldrich) for an hour at 60°C. After washing with distilled water, the slides were stained with Weigert’s iron hematoxylin solution for 10 minutes. The slides were then washed with distilled water and were placed in Biebrich-scarlet acid fuchsin solution for 5 minutes, followed by washing with distilled water. The slides were then placed in an aniline blue solution for 8 minutes. After washing with distilled water, the slides were treated with 0.5% acetic acid for one minute. The slides were then dehydrated in ethanol and treated with xylene for 1 minute. Finally, the slides were mounted with a mounting medium (Dako). Stained slides were analyzed using the Image J software. The fibrotic area (%) was defined as the ratio of Masson’s trichrome-stained area per the total ovary area.