Here we’ve performed a modified HybISS protocol (Gyllborg et al., Nucl. Acids Res., 2020), tailored to zebrafish. We acquire five rounds of widefield images, with different probes in each cycle. In a given cycle, every dot reports a transcript from 100 different genes. Since we have designed the probes used, we know the color code of every one of the 100 transcripts. Therefore, the colors of a dot across the 5 cycles allow us to know which dot corresponds to which gene.