Wild-type zebrafish embryos were raised with or without purified snail extracts from H. aspersa. At 26 hpf, 20 embryos without any obvious deformities were selected for each condition and fixed with 4% (v/v) paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO, USA), dehydrated in 100% (v/v) methanol, and stored at −20 °C. WISH was performed with the vascular probe fli1. The preparation of the probes and WISH in zebrafish embryos was performed as previously described in [33 (link)]. WISH images were taken with a Leica MZ16F stereomicroscope equipped with a DFC 480 digital camera and LAS Leica Imaging software (Leica, Wetzlar, Germany) at 63× magnification.
WISH images were quantified using ImageJ Fiji software, as follows: The region of the embryo tail containing the WISH signal (approximately from the mid-yolk region to the tip of the tail) was selected, and the intensity was measured. An equal area of the tail outside of the stained area was selected to determine the background. The value of the colorimetric signal was then obtained by subtracting the background from the measured intensity.
WISH images were quantified using ImageJ Fiji software, as follows: The region of the embryo tail containing the WISH signal (approximately from the mid-yolk region to the tip of the tail) was selected, and the intensity was measured. An equal area of the tail outside of the stained area was selected to determine the background. The value of the colorimetric signal was then obtained by subtracting the background from the measured intensity.
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