Mouse anti cd63 e 12

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.