Drosophila tracheal cell dissection

The Drosophila line used was reported previously (Best and Leptin, 2020) and carries a recombined btl-Gal4 (Shiga et al., 1996) and UAS-DsRed1 (BDSC 6282) element on the third chromosome, driving expression of DsRed in all tracheal cells. The flies were grown on standard cornmeal-agar medium at 25°C. Wandering third-instar larvae were gently collected from the vial wall using a brush and transferred to a droplet of 4°C Shields and Sang medium on a dissection plate. The larvae were filleted according to standard protocol, exposing the dorsal tracheal system attached to the skin, with all internal organs removed. After confirming that the tissue was still alive by observing the twitching of muscles on the skin, we removed the head and posterior end of the fillet. The remaining sample usually contained completely the five segments A1–A5 (corresponding to tracheal dorsal branch pairs 3–7). This was transferred directly to the high-pressure freezing carrier, prefilled with 20% Ficoll (Sigma; PM70) in Shields and Sang medium.