Biebrich scarlet acid fuschin

Whole gastrocnemius and whole tibialis anterior muscles were removed from both hindlimbs from the implant control group, SFN neurectomy legs, and contralateral control, and BTX injected legs and contralateral control and weighed. Muscle histology was then produced the same as in a previous study^{42 (link)}. Briefly, the gastrocnemius was fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Muscles were cross-sectioned ~0.5 cm from the margins. Sections (5 μm) were placed on Histobond slides (VWR, Radnor, PA, USA), deparaffinized and rehydrated, and stained with Masson’s trichrome using Weigert’s hematoxylin (Sigma-Aldrich, St. Louis, MO, USA), Biebrich scarlet-acid fuschin (Sigma-Aldrich), and aniline blue (Sigma-Aldrich). Coverslips were mounted with xylene-based mounting media and allowed to dry flat before imaging.

Paraffin-embedded tissue was sectioned at 7 μm thickness and stored at room temperature until processing. Slides were deparaffinized in xylene (10 min, twice) followed by 100% ethanol (10 min, twice), then 5 minutes each in 95%, 70% and 50% ethanol. Slides were then rinsed briefly in tap water and diH₂O and fixed in Bouin’s Solution (Sigma, Cat # HT10132) at room temperature overnight. Slides were then rinsed in a container with running tap water until clear, then rinsed once in diH₂O and stained with Biebrich Scarlet-Acid Fuschin (Sigma, Cat # HT151) for 5 min. Rinsing in tap water then diH₂O was repeated, followed by 5 minutes in 5% phosphotungstic/phosphomolybdic acid solution for 5 minutes (Sigma, Cat # HT152 and HT153 respectively). Slides were rinsed in diH₂O then stained for 4 minutes in Aniline Blue solution, then rinsed again in diH₂O, and set in 1% acetic acid for 2 min. Slides were then dehydrated by consecutive two dips in diH₂O, 75%, 95%, and 100% ethanol, then twice in xylene, and mounted using Cytoseal (Richard-Allan Scientific, Cat # 8310–4).