Ti inverted microscope

All whole-mount immunostaining images were collected with a Nikon A1R point scanning confocal with spectral detection and resonant scanner on a Nikon Ti-E inverted microscope equipped with a Plan Apo VC ×20 objective (NA 0.75). Alexa-488, Alexa-594, Alexa-647 fluorophores coupled to secondary antibodies were excited with the 488 nm, 561 nm, and 647 nm laser lines from a Spectral Applied Research LMM-5 laser merge module with solid-state lasers (selected with an AOTF) and collected with a 405/488/561/647 quad dichroic mirror (Chroma). For time-lapse experiments, images were acquired with a Yokagawa CSU-X1 spinning disk confocal on a Nikon Ti inverted microscope equipped with a Plan Apo ×20 objective (NA 0.75) and a Hamamatsu Flash4.0 V3 sCMOS camera. Samples were grown on six-well glass-bottom multiwell plates with no. 1.5 glass (Cellvis, Cat# P06-1.5H-N) and mounted in a OkoLab 37°C, 5% CO₂ cage microscope incubator warmed to 37°C. Images were collected every 15 min using an exposure time of 800 ms. At each timepoint, 30 z-series optical sections were collected with a step size of 2 µm. Multiple-stage positions were collected using a Prior Proscan II motorized stage. Z-series are displayed as maximum z-projections, and gamma, brightness, and contrast were adjusted (identically for compared image sets) using Fiji/ImageJ (Schindelin et al., 2012 (link); https://imagej.net/Fiji).