Rabbit anti shh h 160

Stage 21HH and 24HH legs were homogenised in RIPA buffer (Fisher) containing protease inhibitors, centrifuged, and the supernatant is collected. Protein concentration was estimated using a DC Protein Assay kit (Bio-Rad). Recombinant mouse SHH N-terminus protein (R&D Systems) was used as a positive control. Protein samples were loaded as individual limbs per lane. Proteins were separated by electrophoresis using pre-cast 12% gels (Invitrogen), transferred to nitrocellulose membranes by standard procedures. Membranes were blocked in Odyssey Blocking Buffer (Licor), incubated with 1:100 rabbit anti-SHH H-160 (Santa Cruz, sc9024) 1:2500 mouse anti-γ-tubulin (Sigma, T5326) 4 °C overnight, followed by goat anti-rabbit 680CW (Licor, 926-32221) goat anti-mouse 800CW (Licor, 926-32210) for 1 hour. Membranes were dried and signal detected using an Odyssey Infrared Imager (Licor). Bands were quantified using Image Studio software, and normalised to γ-tubulin protein.

Cell fixation and staining were performed using MEM-fixation protocols (Hall and Ogden, 2018 (link)). The following antibodies and dilutions were used: rabbit anti-SHH (H-160) (1:100; Santa Cruz), mouse anti-GFP (4B10) (1:500; CST), rat anti-HA (1:250; Roche), mouse anti-CD63 (E-12) (1:100; Santa Cruz), rabbit anti-Myc-Tag (2272) (1:400; CST). Secondary antibodies (Jackson ImmunoResearch and Invitrogen) were used at a 1:1000 dilution. For additional information please refer to Appendix 1—key resources table. Lipid raft staining was performed using Cholera Toxin Subunit B (Recombinant) (CTX), Alexa Fluor 488 Conjugate (Invitrogen). CTX was dissolved in chilled PBS to a final concentration of 1.0 mg/mL. CTX was incubated with cells for 20 min at 4°C to prevent endocytosis. Cells were rinsed three times in chilled PBS prior to MEM-fixation. Extracellular staining was performed by diluting antibodies in 4°C PBS supplemented with 5% normal goat serum. Antibody solutions were then incubated for 30 min on live cells on ice to prevent endocytosis. Cells were rinsed three times in chilled PBS prior to MEM-fixation. Microscopy images were taken with a TCS SP8 STED 3X confocal microscope (Leica) for fixed and live cell imaging.