Probes for DNA-FISH were prepared using the FISH Tag™ DNA Multicolor Kit (Thermo Fisher) following kit instructions. The BAC DKEY-69C19 was used as a template for DNA-FISH probe production, corresponding to 214 kb of the mir430 locus on chromosome 4.
Embryos were fixed at the developmental stage of interest using 4% PFA. Animal caps were isolated and a series of gradient washes was used to equilibrate the samples with hybridization buffer [50% formamide, 4x SSC, 100 mM NaPO4 pH 7.0, 0.1% Tween-20]. Embryonic DNA was denatured by incubation of samples at 70 °C for 15 min before application of probe and incubation overnight at 37 °C. Unbound probe was washed from the samples using a hybridization buffer: PBS-T gradient. Samples were counterstained with DAPI prior to imaging on a Zeiss LSM 880 with Fast Airyscan, with a 100 × 1.48 numerical aperture objective lens. 50–100 optical sections (130 nm thickness) were acquired of each nucleus. Acquired images were processed using Zen Black software (Zeiss).
Embryos were fixed at the developmental stage of interest using 4% PFA. Animal caps were isolated and a series of gradient washes was used to equilibrate the samples with hybridization buffer [50% formamide, 4x SSC, 100 mM NaPO4 pH 7.0, 0.1% Tween-20]. Embryonic DNA was denatured by incubation of samples at 70 °C for 15 min before application of probe and incubation overnight at 37 °C. Unbound probe was washed from the samples using a hybridization buffer: PBS-T gradient. Samples were counterstained with DAPI prior to imaging on a Zeiss LSM 880 with Fast Airyscan, with a 100 × 1.48 numerical aperture objective lens. 50–100 optical sections (130 nm thickness) were acquired of each nucleus. Acquired images were processed using Zen Black software (Zeiss).