Inverted a1r spectral microscope

Transgenic fluorescent embryos were embedded in 1% agarose in a glass-bottom dish. Immunofluorescence double staining was performed as described previously^{57 (link)}, with chicken anti-GFP (1:400; Life Technologies) and rabbit anti-phospho-histone 3 (pH3) antibodies (1:250; Abcam). AlexaFluor488-conjugated anti-chicken secondary antibody (1:1000; Life Technologies) and AlexaFluor594-conjugated anti-rabbit secondary antibody (1:1000; Life Technologies) were subsequently utilized to reveal primary antibodies. Confocal imaging was performed using a Nikon inverted A1r spectral microscope.

For time-lapse imaging, Tg(kdrl:mCherry;cmyb:GFP) embryos were anaesthetized with 0.03% Tricaine (Sigma), and embedded in 1% agarose in a 60 mm glass-bottom dish. The embryos were imaged at 28.5 °C with a confocal Nikon inverted A1r spectral microscope. Scanning with 20x water immersion objective, z-stalks were acquired with a step size of 7 μm with an interval of 7 min for 6 h in the control and ifi30-morphants starting at ∼42 hpf, and an interval of 10 min for 7 h in non-treated and during heptanol treatment starting at ∼41 hpf. The analysis of cmyb:GFP⁺ cells in all experiments was performed using IMARIS image software (version 9.7.2). The 3D + t acquisitions were manually oriented to have the CHT horizonal. Then, when required, the presence of a 3D translational drift was corrected by detecting structures that are morphologically stable (therefore not intrinsically moving during the time course). Subsequently, a background subtraction was performed on the GFP channel. Finally, for display purposes, maximum intensity projections were used and the intensity of each channel of the images was set in order to saturate the bottom 1% and the top 0.1% of all pixel values. Movies were recorded using Lanczos’ resampling with a kernel of size 3 and quantized over 8 bits per channel afterwards.