Weigert s iron hematoxylin working solution

Trichrome reagents were obtained from Electron Microscopy Sciences, unless otherwise stated. After heating the paraffin-embedded slides of excised wounds at 60 °C, the slides were deparaffinized with citrosol twice (2 × 3 min) and rehydrated through 100% ethanol (×2), 95% ethanol, and 70% ethanol (3 min each) followed by washing in distilled water. Then, the slides were refixed in Bouin’s solution overnight at room temperature. After 10 min of rinsing in running tap water, the tissues were stained in Weigert’s iron hematoxylin working solution (Sigma-Aldrich) for 10 min, rinsed in running warm tap water for 10 min, and briefly washed in distilled water. The slides were stained in Biebrich scarlet-acid fuchsin solution for 15 min, washed in distilled water, and differentiated in phosphomolybdic–phosphotungstic acid solution for 15 min. The slides were then transferred directly to aniline blue solution and stained for 10 min, followed by a brief rinse in distilled water. In the next step, the sections were differentiated in 1% acetic acid solution for 5 min, washed in distilled water, and dehydrated very quickly through 95% ethanol and 100% ethanol (30 s each) and then cleared in citrosol (1 min). Finally, the sections were mounted with a xylene-based mounting medium (Triangle Biomedical Sciences).