Plan apochromat 63 1.4 na oil objective lens

Xenopus embryos were mounted between cover glass and submerged in 1/3x MMR at stage 22 or 23, and then were imaged immediately. Live images were captured with a Zeiss LSM700 laser scanning confocal microscope using a plan-apochromat 63 × 1.4 NA oil objective lens (Zeiss) or with Nikon eclipse Ti confocal microscope with a 63×/1.4 oil immersion objective. For FRAP experiments, a region of interest (ROI) was defined as a 1.75 µm X 1.75 µm box. ROIs were bleached using 50% laser power of a 488 nm laser and a 0.64 µsec pixel dwell time. Fluorescence recovery was recorded at ~ 0.20 s intervals for up to 300 frames. Bleach correction and FRAP curve-fitting was carried out using a custom python script (modified from http://imagej.net/Analyze_FRAP_movies_with_a_Jython_script). For colocalization analysis, z- stack images were captured from at least 15 cells from at least five different embryos. Z stacks were split to single sections and ROI for DynAPs was defined in red channel (mCherry) of each section using ImageJ selection tool. The analysis was carried out using Fiji coloc2 plugin.
Intensity of dynein subunits and length of axonemes were measured using Fiji. Plots were generated using PRISM eight and the ggplot2 package in R. One-way ANOVA and Tukey’s Honest Significant Difference (HSD) test and Welch Two Sample t-test were performed in R.

Embryos expressing fluorescent proteins were fixed at stage 26 with 1x MEMFA (0.1 M MOPS, 2 mM EGTA, 1 mM MgSO₄, 3.7% formaldehyde, pH7.4) for 40 min at stage 26, washed with PBS and then imaged. For live images, Xenopus embryos were mounted between cover glass and submerged in 1/3x MMR at stage 25–28. Imaging was performed on a Zeiss LSM700 laser scanning confocal microscope using a plan-apochromat 63 × 1.4 NA oil objective lens (Zeiss) or Nikon eclipse Ti confocal microscope with a 63 × 1.4 oil immersion objective. For FRAP experiments, a region of interest (ROI) was defined for full bleach experiments as a 1.75 µm× 1.75 µm box and for half-bleach experiments as a 0.8 µm× 0.4 µm box. ROIs were bleached using 50% laser power of a 488 nm laser and a 0.64 µs pixel dwell time. Fluorescence recovery was recorded at ~0.20 s intervals for up to 300 frames. Bleach correction and normalization was carried out using a custom python script (modified from http://imagej.net/Analyze_FRAP_movies_with_a_Jython_script). Plots were generated using the ggplot2 package in R. 3D projections were generated in Fiji or IMARIS.