Plan apo 63

Cells were imaged in RPMI 1640 medium at 37°C. Images were acquired with a spinning disk confocal system (spinning disc CSU22) fitted on an eclipse Ti microscope (Nikon) with oil immersion Plan-Apo 63× NA 1.4 objective and a charge coupled device camera (EM-CCD C-9100-2). Acquisition and processing of images was performed with Volocity Software (Improvision). Cells were seeded on glass-bottomed dishes (35 mm; Ibidi) at a density of 2 × 10⁵ and incubated for 20 h before the start of the experiment. TIRF imaging was performed with an iLAS TIRF unit from Visitron Systems fitted on an eclipse Ti microscope (Nikon) with oil immersion Plan-Apo 63× NA 1.45 and Plan-Apo 100× NA 1.49. To correct for drift of the microscope stage, TetraSpec Microspheres (0.1 µm, fluorescent blue/green/orange/dark red; Thermo Fisher Scientific) were used.

Confocal images were acquired on an LSM 510 (ZEISS) with Plan-Apo 63× (1.40 NA) oil-immersion objective, A1R-Si+ (Nikon) with Plan-Apo 60× (1.40 NA) oil-immersion objective, or Fluoview FV1000 (Olympus) with Plan-Apo 60× (1.42 NA) oil-immersion objective. Brightness and contrast were adjusted using the Fiji distribution of ImageJ (Schindelin et al., 2012 (link)). For analysis of Cacophony puncta intensity, all genotypes were stained together and imaged with identical settings. For consistency, analysis was limited to NMJ 4 in segments A2 and A3. To measure Cac-GFP and HRP intensity, nonsynaptic structures including axons were removed from the images using freehand selection and fill. z-stacks were flattened using the Maximum Intensity Z-projection function. Channels were separated and a threshold was applied to remove irrelevant lower-intensity pixels. Separation of individual puncta was facilitated by the Find Maxima tool in Fiji. Intensity data were collected using the Analyze Particles tool.