Fig. 2

Qualitative evaluation of natterin-like gene expression by whole-mount in situ Hybridization (WISH). Larvae after 24 h post-fertilization (hpf) were fixed, dehydrated, permeabilized and hybridized with the DIG-labeled probe, complementing the natterin-like RNA sequence (Qiagen, #339500 LCD0168623-BKG). Probe hybridization was determined by binding the AP-labeled anti-DIG antibody (#11093274910, Roche Diagnostics) and revealed through chromogenic reaction by NBT/BCIP solution. The images were obtained in the stereomicroscope STEREO LUMAR-V-12 Carl Zeiss. The picture snippets indicate the area of the larva’s body where the blue-purple precipitate (indicating gene product) can be observed in the non-depleted wild-type (WT) larvae (left), indicative of the presence of the natterin-like transcripts; and in the depleted (KO) larvae by CRISPR/Cas9 system (crRNA natterin-like WD07479944, Sigma) (right). The embryos body changed the shape due to long-lasting high temperature incubation in conical-bottom microtubes, not representing developmental malformations