Zebrafish embryo maintenance, RPL5 morpholino (MO) microinjection, and hemoglobin staining

Wild-type zebrafish (Danio rerio; AB type) were bred and maintained under standard library conditions. Zebrafish embryos were kept in a 28.5 °C incubator, and the embryonic stages evaluated in this study have been previously described [44, 45]. The RPL5 MO (5-ACCCATTTTGTGATCGTTTGTTCTC-3), control MO (5- ACCCGTTTCGTAATCGTCTGTGCTC-3) and P53 MO (5- GCGCCATTGCTTTGCAAGAATTG-3) were obtained from Gene-Tools, LLC (Philomath, OR, USA). Zebrafish embryos at the one-cell stage were injected with the MOs using a microinjector (WPI SYS-PV830). Based on our initial trials, 0.25 ng RPL5MO and control MO was chosen as the optimal concentration. Injected embryos were grown at 28.5 °C and observed under a microscope. The effectiveness of translational inhibition by RPL5 MO was tested in vivo using the RPL5:egfp green fluorescent fusion protein under fluorescence microscopy. The O-dianisidine (Sigma) staining protocols were performed as previously described [23] [24] and images were collected using an Olympus microscope with a digital camera (OLYMPUS IX71) and imported into Adobe Photoshop CS2 9.0.2 for orientation and figure preparation.