P3130

Embryo and pupal antenna immunohistochemistry was carried out according to (zur Lage et al., 2018 (link)). The fixing and staining of Drosophila testis was described in Sitaram et al., 2014 (Sitaram et al., 2014 ). The following primary antibodies were used: rabbit anti-GFP antibody (1:500, Life Technologies, A11122), mouse anti-Futsch antibody (1:200, Developmental Studies Hybridoma Bank, 22C10-s), mouse anti-acetylated tubulin (1:1000, Sigma, T6793) and rabbit anti-Dnah5 antibody (1:2000, see below) and the secondary antibodies were goat anti-Rabbit antibody (1:500, Alexa Fluor 488, Life Technologies, A11008) and goat anti-Mouse antibody (1:500, Alexa Fluor 568, Life Technologies, A11019). DNA in adult testes was stained with To-Pro-3 (1:1000, Life Technologies, T3605) solution in the dark for 15 min. After several washes, the samples were mounted on slides with 85% glycerol and 2.5% propyl gallate (Sigma-Aldrich, P3130). The slides were imaged using a Zeiss LSM-5 PASCAL/Axioskop 2 confocal microscope and processed with Fiji.

Approximately 0.5 × 10⁶ cells per well were seeded in a 6-well plate containing glass coverslips and allowed to incubate in complete medium (DMEM containing 10% FBS, 1% penicillin–streptomycin) for 24 h prior to cilia induction by culturing cells in starvation medium (0.5% FBS) for 48 h followed by three times washing in ice-cold phosphate-buffered saline (PBS). They were then fixed in 4% paraformaldehyde for 15 min, washed three times in PBS, permeabilized with 1% Triton-X-100 in PBS for 15 min, and incubated in blocking solution [PBS containing 3% bovine serum albumin (BSA)] for 30 min. The cells were incubated with primary antibodies overnight at 4 °C and washed three times in blocking solution. Secondary antibodies were added for 45 min at room temperature followed by washing for 5 min in blocking solution. They were then incubated with 0.5 μg ml⁻¹ 4′, 6-diamidino-2-phenylindole for 30 s to stain DNA, washed three times in PBS and mounted with an anti-fading mounting gel containing N-propyl gallate (Sigma-Aldrich #P3130). All procedures were performed at room temperature. Confocal microscopy was performed using an Olympus Fluoview 1000 FV. Adjustments to brightness and contrast were minimal and applied to the whole image. Z-stacked images were Z-projected and Fiji software (http://fiji.sc) was used for measuring cilia length.