Lipid Raft and Ret Patching Protocol

Lipid raft patching was induced as described by Fra et al. and Peter et al. with slight modifications [29 (link),30 (link)]. Briefly, for all fluorescence microscopy experiments, the cells were attached to 35 mm poly-l-lysine-coated dishes at a density of 2 × 10⁵. Cholera toxin subunit B (CT-B) labeled with Alexa Fluor 594 (Molecular Probes Inc., Eugene, OR, USA) was used at 10 mg/mL in PBS to label ganglioside GM1, which selectively partitions into lipid rafts. An anti-CT-B antibody (1:200 in PBS; Molecular Probes Inc.) was used to crosslink the CT-B-labeled lipid rafts into distinct patches on the plasma membrane. The cells were then incubated for 10 min and 15 min at 4 °C. The presence of lipid raft aggregation or patching was confirmed after CT-B and anti-CT-B labeling. For fixation, the cells were directly treated with 4% paraformaldehyde in PBS for 15 min.
Ret patching was induced in the same manner. The cells were incubated with a primary anti-Ret antibody (1:1000, Abcam) overnight at 4 °C, and this was followed by incubation with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes Inc.) for 2 h at room temperature. Confocal microscopy (FV10-ASW, Olympus, Tokyo, Japan) was performed with a 60× oil immersion objective, using laser excitation at 350, 488, and 594 nm.