Weigert s iron hematoxylin set

Dewaxed hydrated paraffin-embedded tissue sections were refixed in Bouin’s solution (HT10132; Sigma-Aldrich) and then stained with Weigert’s Iron Hematoxylin Set (HT10–79; Sigma-Aldrich) and Masson’s Trichrome Staining Kit (HT15; Sigma-Aldrich) for red muscle fibers and blue collagen, as described by the manufacturer.

Sections from WT and miR-205^−/− transplanted outgrowths were subjected to a series of steps of deparaffinization and rehydration then mordant in Bouin’s Solution (Millipore Sigma, St. Louis, Missouri, www.emdmillipore.com/US/en/home.html) at room temperature overnight to intensify the final coloration of the tissue. Weigert’s Iron Hematoxylin Set (Sigma) was used to stain nuclei. Sections were then put in Biebrich Scarlet-Acid Fuchsin from Accustain Trichrome Stain Kit (Sigma) for 5 minutes to stain cytoplasm and muscle red. Phosphomolyb-dic/Phosphotungstic Acid Solution (5 minutes) and Aniline Blue (5 minutes) from Accustain Trichrome Stain Kit (sigma) were used to develop collagen blue staining. Upon detection, sections were rinsed in 1% acetic acid solution for 5 minutes, dehydrated, and mounted using Permount (Fisher Scientific).