Drosophila Thorax Cryosectioning and Staining

Head, wings and abdomen were removed from one day old w¹¹¹⁸ flies and thoraxes were fixed overnight at 4°C in 4% PFA. For 30–90 hr APF samples, pupae were freed from the pupal case, poked 3–5 times with an insect pin in the abdomen and fixed overnight at 4°C in 4% PFA. Thoraxes or pupae were then sunk in 30% sucrose in 0.5% PBS-T overnight at 4°C on a nutator. Thoraxes or pupae were embedded in Tissue-Tek O.C.T. (Sakura Finetek) in plastic moulds (#4566, Sakura Finetek) and frozen on dry ice. Blocks were sectioned at 30 µm on a cryostat (Microm vacutome). Sections were collected on glass slides coated with 1% gelatin +0.44 μM chromium potassium sulfate dodecahydrate to facilitate tissue adherence. Slides were post-fixed for 1 min. in 4% PFA in 0.5% PBS-T at RT, washed in 0.5% PBS-T, incubated with rhodamine-phalloidin for 2 hr at RT, washed three times in 0.5% PBS-T and mounted in Fluoroshield with DAPI (#F6057, Sigma).