Csu x 1 spinning disc

Confocal imaging was performed at the Nikon Imaging Center at the Whitehead Institute for Biomedical Research using a spinning disc system (Krakowiak et al., 2018 (link)) with the following parameters: Nikon Ti-E base, 1.49 NA, 100× objective, Yokogawa CSU-X1 spinning disc, Andor iXon 897E EM charge-coupled device camera, and MetaMorph acquisition software. Cells were grown and indicated fluorophores were imaged in SDC media, and heat shock was performed on live cells from 25°C to 39°C using an objective heater (Chowdhary et al., 2019 (link)). Images were autocontrasted using ImageJ, and single z-slices are shown.

The ANSs were dissected as above and maintained in chilled locust saline until fixation (up to 60 min). For the purpose of immunostaining and histology, samples were prepared from a section of the dissected preparation (comprising the second abdominal ganglion and a section of the paired connectives), and thoroughly cleaned of all non-nervous tissue.
Samples were fixed in 4% PFA for 16 h at 4°C, then washed in PBS and permeabilized with ice-cold MeOH at −20 °C for 5 min, blocked, and further permeabilized with 10% fetal bovine serum and 1% Triton for 2h. Samples were agitated and actin was stained with Fluorescein Phalloidin (Invitrogen) at 10 μg/mL for 1h. Finally, samples were mounted with VectaShield (Vector Laboratories). Cover slides were sealed with nail polish until use.
Confocal images were captured using a Nikon Ti microscope equipped with a Yokogawa CSU X-1 spinning disc and an Andor iXon897 EMCCD camera controlled by Andor IQ3 software. Image analysis was performed using FIJI ImageJ V.2.0.0.

Imaging experiments were performed using either a 1) Nikon TI inverted microscope with NIS Element Software (Nikon), equipped with a perfect focus system, a Yokagawa CSU-X1 spinning disc, an iXon Ultra 897 EM-CCD camera (Andor), or 2) a Nikon TI2 inverted microscope with NIS Element Software, equipped with a perfect focus system, a Yokagawa CSU-X1 spinning disc and an Prime 95B sCMOS camera (Photometrics). Both microscopes were equipped with a temperature-controlled hood. For experiments involving long-term time-lapse analysis (> 3 hr) a 60x 1.40 NA oil-immersion objective was used, while a 100x 1.49 NA oil-immersion objective was used for short-term analyses. A 100x 1.49 NA oil-immersion objective was used for experiments using 2D organoid cells. The siRNA screen (Figure 6B, 6C, S6B, S6H, S6J, and S6O) was performed using a 40x 0.95 NA air objective.