Uplansapo 60 na1.35 objective

Cells were fixed with 4% formaldehyde for 10 min, permeabilized with 0.1% Triton X100 for 5 min, and blocked with 2% BSA for 1 h. Cells were then incubated with primary antibodies to vinculin (Sigma V9131) or paxillin (Abcam 32084) for 2 h, labeled with secondary antibodies (30 min), and imaged via fluorescence microscopy (Olympus IX83, UPlanSApo 60×/NA1.35 objective).

Confocal microscopy was performed on an FV1000-BX61 laser-scanning confocal microscope using an UPlanSApo 60×/NA 1.35 objective (all Olympus). Movies were captured using the FV1000-IX81 microscope using a PlanApo N 60×/NA 1.42 objective (all Olympus). Macrochaetes were imaged for all experiments. z-stack image generation and brightness and contrast adjustment were performed using ImageJ (NIH) without any nonlinear adjustments. Gaussian filter was applied to generate still images from time-lapse imaging of S2 cells.
The ‘tip index’ was determined as follows (see also supplementary material Fig. S1). A line scan was performed from the base of the bristle to the distal tip to obtain a plot profile using ImageJ. Subsequent analyses were performed using Excel (Microsoft). The maximum intensity (100% intensity) and bristle length (Position[Max]) were determined from the line scan, and pixels that exceeded 50% intensity were identified. The tip index was defined as the relative position of the pixels that exceeded 50% intensity along the proximal-distal axis of the bristle; the full bristle length was defined as 100. Statistical analyses (Student's t-test) were performed using Excel.