Agar 100

Wing-discs from 3rd instar larvae were prepared as described previously (Stevens et al., 2010). Briefly, the wing discs were dissected in PBS and then fixed in 2.5% glutaraldehyde, 4% paraformaldehyde, and 0.1% tannic acid (from a freshly prepared 10% stock) in 0.1 M PIPES buffer (pH 7.2) for 1 h (up to 2 h) at RT and left overnight in the fridge at 4°C. Samples were then washed twice in 0.1 M PIPES, followed by one wash in 50 mM glycine in 0.1 M PIPES to quench free aldehydes, and then another wash in 0.1 M PIPES. Samples were then post-fixed in 1% OsO₄ for 2 h at RT, followed by extensive washing in distilled water. Samples were stained with 0.5% uranyl acetate overnight at 4°C, washed in distilled water, dehydrated in an ethanol series and embedded in Agar100 (Agar Scientific). Blocks were polymerized at 50°C for 24–42 h. Semi-thin serial sections (100 nm) were obtained in a Leica EM UC7 ultramicrotome (Leica Microsystems) and stained in lead citrate. Images of centrioles in longitudinal orientation were taken on a TECNAI T12 transmission microscope (FEI) at 13,000X magnification to measure centriole length from the wing discs. The length of the MT doublets within the electron-dense area was measured using the line tool in Fiji (ImageJ).

For electron microscopy, lung tissue was fixed in 2.5% (wt/vol) glutaraldehyde in 0.1 M cacodylate buffer at pH 7.3 for 3 to 4 hours. After dehydration, the tissue was embedded in resin (AGAR-100, Agar Scientific). Ultrathin lung sections (90 nm) were contrasted with uranyl acetate/lead citrate and studied with a Zeiss EM 900 electron microscope. Mitochondrial cristae were identified on micrographs made with a ThermoFisher (formerly, FEI) Tecnai G2 20 electron microscope, operated at 120 kV, with an Ametek (formerly, Gatan) Ultrascan 1000 camera.