Axiozoom v16 epifluorescence microscope

For examining X. tropicalis eye phenotypes, injected embryos were allowed to develop to stage 42 and scored for eye defects. The percentages of normal and reduced phenotypes were calculated for injected embryos obtained from multiple independent experiments, and chi-squared tests were performed to compare the phenotypes in different treatment groups. Images of eyes (bright-field) were taken with a Zeiss Axiozoom.v16 epifluorescence microscope.

Wild-type X. tropicalis adults (male and female) were purchased from the NASCO. Methods involving live animals were carried out in accordance with the guidelines and regulations approved and enforced by the Institutional Animal Care and Use Committees at the University of Delaware. Morpholinos (MOs) were designed using Gene Tools, LLC, OR. The Hspb1 MO is a 25-mer with the sequence 5′ GTA TTC TGC GTT CTG ACA TTT TC 3′. The control MO is the standard control MO obtained from Gene Tools with the sequence 5′ CCT CTT ACC TCA GTT ACA ATT TAT A 3′. Embryos were collected and injected with a PLI-100A microinjector (Harvard Apparatus) as described previously (106 (link)). Control or HSPB1 morpholino (1.5 ng per blastomere) was injected into a single dorsal-animal blastomere at the eight-cell stage; Alexa Fluor 568 dextran (Invitrogen #D22912) was co-injected as a lineage tracer. For the rescue experiments, mouse Hspb1 mRNA was generated by in vitro transcription as described (107 (link)) and co-injected with the HSPB1 morpholino (50 pg mRNA per blastomere). Injected embryos were cultured in 0.1X MBS to desired stages, and eye phenotypes were observed using a Zeiss Axiozoom.v16 epifluorescence microscope.