The 2.5D invadopodia localization assay was adapted from Magalhaes et al. (2011) (link). In brief, growth factor–reduced Matrigel (2.5 mg/ml; BD) was mixed with 0.2 mg/ml Alexa Fluor 405 gelatin (1:20 dilution) and applied to the top chamber of a 1.0-µm transwell support (Corning). Excess Matrigel/gelatin solution was removed, and both chambers were allowed to equilibrate in plain DMEM at 37°C for 1 h. After equilibration, the bottom chamber was filled with 750 µl of DMEM/10% FBS. 30,000 cells were resuspended in 200 µl of DMEM/0.5% FBS, plated in the top chamber, and allowed to invade for 24 h. Cells were fixed in 3.7% paraformaldehyde and immunofluorescently labeled for cortactin and pY402-Pyk2. Membranes were imaged using an inverted laser-scanning confocal microscope (LSM780; 60× 1.4 NA with oil objective with ZEN black edition acquisition software; ZEISS). 3D invadopodia images were reconstructed using Imaris 8.0.0 (Bitplane).