Lsm 7 microscope

Transgenic fish were generated by microinjection of test constructs together with 50–100 pg of Tol2 transposase mRNA into fertilized 1-cell-stage embryos [42 (link)]. Three day-post-fertilization larvae were screened for mCherry fluorescence in the majority of heart cells. Larvae were anesthetized and larval fin folds were amputated at a position immediately caudal to the end of the notochord using a 22.5 degree microknife (FST) and PDMS cutting surface. Fish were then embedded in 1.5% low-melt agarose and imaged on Zeiss LSM 7 microscope with the Plan-Neofluar 10× (numerical aperture 0.30) objective. Time-lapse images were captured at a frequency of 1 z-stack every 10, 7, or 5 min for up to 12 h. Adult fins were amputated using a razor blade approximately halfway along the length of the fin and maintained at 28 °C. Adult fish were imaged on either a Nikon SMZ1500 outfitted with the Nightsea Stereomicroscope Fluorescence Adapter system and imaged with a Nikon D80 camera or on a Zeiss Stemi SV11 Apo and imaged with a Zeiss AxioCam HRc camera.

neomycin sulfate (Sigma) was dissolved in zebrafish embryo water to a stock concentration of 6 mM and then diluted to a working concentration of 50 μM [43 (link)]. Transgenic embryos were generated and screened for heart expression as above, then 5-day post fertilization larvae were incubated in neomycin for 1 h at 29 °C, rinsed in embryo water, and allowed to recover for approximately 0.5 h before initial imaging, then imaged at 24-h post injury on a Zeiss LSM 7 microscope with the Plan-Apochromat 20x (numerical aperture 0.8) objective. neomycin injury was confirmed by equivalently treating wild type larva and observing loss of DASPEI (2-[4-(dimethylamino)styryl]-l-ethyl-pyridinium iodide) staining [43 (link)].

For MBP immunofluorescence 8 dpf zebrafish larvae were fixed in 4%PFA/PBS, overnight at 4ºC. Larval brains were dissected and incubated with rabbit anti-MBP (Lyons et al., 2005 (link)). Anti- rabbit Alexa Fluor568 was used at 1/1000 (A-11011, ThermoFisher). Embryos were mounted in DAPI Fluoromount-G (SouthernBiotech). Transgenic zebrafish embryos were mounted in 1.5% low melting point agarose in distilled water. Images were captured using a Zeiss LSM 5 Pascal or Zeiss LSM 7 microscope. Objectives used were Plan-Neofluar 10× (numerical aperture 0.30) and 20× (numerical aperture 0.75). All fish were genotyped after imaging and analysis.