Tcs sp8 wll system

AO staining was performed as described^{27 (link)}, with some modifications. mEPCs cultured in 29 mm glass-bottom dishes (Cellvis, D29-14-1.5-N) to day 10 post serum starvation were incubated with 100 nM siR-tubulin (SPIROCHROME, CY-SC002) for 1 h at 37 °C, followed by fixation with 4% PFA in PBS for 15 min at 4 °C. After treated with PBS (mock) or 100 μg/ml RNase A (ThermoFisher Scientific, 12091021) in PBS for 30 min at 37 °C, the cells were incubated with 20 μg/ml AO (Sigma–Aldrich, 318337) in staining buffer (37 mM citric acid, 126 mM Na₂HPO₄, 150 mM NaCl, 1 mM EDTA, pH 3.8) for 15 min at 4 °C. After staining, the cells were mounted in Prolong Diamond Antifade Mountant (ThermoFisher Scientific, P36970) and immediately imaged on a Leica TCS SP8 WLL system with 561 excitation/ 650 emission (for RNA fluorescence), 488 excitation/ 525 emission (for DNA fluorescence), and 650 excitation/690 emission (for siR-tubulin fluorescence) filter sets. As siR-tubulin staining of multicilia tended to fade rapidly if permeabolized with Triton X-100, we chose to directly treat the cells with RNase without detergent permeabilization. We confirmed that the RNase treatment was effective (Supplementary Fig. 1b).

Confocal microscopy images were taken on the Leica TCS SP8 WLL system with a ×63/1.4 oil immersion objective. Optical sections were captured at 0.5 μm intervals and z-stack images were obtained with maximum-intensity projections. 3D-SIM super-resolution images were acquired using a DeltaVision OMX SR Imaging System (GE Healthcare) equipped with 4 sCMOS (scientific Complementary metal–oxide–semiconductor) cameras (Pco.edge) and a PlanApo ×60/1.42 oil objective (Olympus). Immersion oil with refractive index of 1.518 (Cargille) was used to minimize spherical aberrations. Optical sections were acquired at 125 nm z-steps. Raw OMX data were analyzed in SoftWoRX 7.0 software (GE Healthcare) with the following procedure: OMX SI Reconstructed using Channel-specific Wiener filter (0.002 for 488, 0.004 for 568, 0.002 for 647), OMX Align Image and Quick Projection (maximum-intensity projection).
As cell bodies are highly abundant in RNA and components of translational machineries, z-projected images from multicilia-containing z-stacks were presented to avoid the interference of fluorescent signals from cell bodies, unless the influence was neglectable or otherwise mentioned.