Glycerol mounting media

After limb amputation, the samples were placed into freshly prepared 4% paraformaldehyde (PFA) and fixed for 24 h (or 48 h for RNAscope) at +4 °C on a roller. Samples were subsequently cryopreserved in 30% sucrose (VWR C27480) overnight at +4 °C, embedded in either gelatin³⁴ or OCT media (HistoLab 45830) and sections cut between 10 µm to 40 µm on a cryostat (Microm). Mouse tissue was processed identically; all mouse tissue samples were embedded in O.C.T. only. If needed, sections were stored at −20 °C (or −80 °C for RNAscope) after drying or processed immediately after sectioning. For imaging, washed (15 min in PBS) slides with sections were mounted with 87% glycerol mounting media (Merck).

Frozen samples were sectioned at 18–30 µm depending on specific experiment. If needed, sections were stored at −20°C after drying 1 hr at room temperature, or processed immediately after sectioning. Primary antibodies used were: chicken anti-GFP (Abcam, 1:500, ab13970), rabbit anti-SOX9 (Sigma Aldrich, 1:1000, HPA001758), sheep anti-ERBB3 (RnD Systems, 1:500, AF4518). For detection of above-mentioned primary antibodies, we utilized 405, 488, 555 or 647-conjugated Alexa secondary antibodies produced in donkey (Invitrogen, 1:1000). Slices were mounted with 87% glycerol mounting media (Merck).