All embryos were dechorionated in bleach (49 (link)) prior to being mounted ventral-side-up on double-sided tape (Scotch) in a minimal volume of Voltalef oil (VWR). All imaging of embryos was carried out on an UltraView Spinning Disk System (Perkin Elmer) using a 40x UplanSApo oil immersion objective lens (NA 1.3). The ventral region (most medial body segments) of embryos was imaged from the embryonic surface to a depth of 20µm, with z-slices spaced every 1µm. For lysotracker experiments requiring staining of live embryos, stage 15 dechorionated embryos were selected and transferred to a 50:50 mixture of peroxide-free heptane (VWR) and 10μM lysotracker red (Thermofisher) in PBS (Oxoid) in a glass vial, which was shaken in the dark for 30 minutes. Embryos were then transferred into a Watchmaker’s glass containing Halocarbon oil 700 (Sigma), before being mounted as described above.
Embryos requiring fixation and immunostaining were fixed and stained as previously described (17 (link)). For Fascin staining, embryos were treated with a mouse anti-Fascin primary antibody (sn7c; Developmental Studies Hybridoma Bank; used at 1:500), with Alexa fluor 568 goat anti-mouse used as a secondary antibody (A11031, Life Technologies; 1:200).
Embryos requiring fixation and immunostaining were fixed and stained as previously described (17 (link)). For Fascin staining, embryos were treated with a mouse anti-Fascin primary antibody (sn7c; Developmental Studies Hybridoma Bank; used at 1:500), with Alexa fluor 568 goat anti-mouse used as a secondary antibody (A11031, Life Technologies; 1:200).
Free full text: Click here
