Poly lysine microscope slides

Following the 14 days culture period, constructs were fixed using methanol and acetone at increasing concentrations. For C₂C₁₂ and HDMC constructs, the actin cytoskeleton was identified using Rhodamine-Phalloidin (1:500, Thermo-Fisher, UK) and nuclei were stained using DAPI (1:2,000, Thermo-Fisher, UK) in Tris-Buffered Saline (TBS) for 2 h. Slides were then washed (3 × 15 min, TBS) and transferred to poly-lysine microscope slides (Thermo-Fisher, UK) and mounted using Fluoromount™ mounting medium (Sigma-Aldrich, UK). For cryosections of HDMC constructs, sections were stained for Desmin to show myogenic cells and DAPI. Briefly sections were incubated overnight with anti-desmin antibody (1:200, Dako, UK, clone D33) in blocking solution (Tris-buffered saline mixed with polysorbate 20, TBST, 5% Goat serum). Samples were washed (3 × 15 min, TBS) and incubated for 1 h with secondary fluorescent antibody (1:500, Invitrogen, UK, Alexa Fluor™ 588 goat anti-mouse) and DAPI (1:2,000) in blocking solution. Slides were then washed (3 × 15 min, TBS) and mounted as previously described.

Following decalcification, tissues were trimmed, washed in running tap water, and soaked in deionized water for 30 min each. Then the specimens were processed for dehydration in ethanol and clearing in xylol, before infiltration and embedding in molten paraffin wax at 65 °C. For more details, refer to Table S3. For embedding, the samples were oriented with the external malleolus down for the right paw or the internal malleolus down for the left paw so that the soles of the paws were perpendicular to the cutting face.
After paraffin embedding, sagittal sections were cut at 5 μm with a Slee Cut 5062 rotary microtome (Slee Medical GmbH, Nieder-Olm, Germany). Paraffin ribbons at the middle of the ankle and tarsal joints were flattened in a water bath at 40 °C and collected onto polylysine microscope slides (Thermo Scientific) before drying at 45 °C overnight. For more details, refer to Fig. S2 in the Supplementary information. The paraffin sections were ready for histological staining.