Embryos were dechorionated and glued to a coverslip as above, dehydrated for 10–15 min, and covered with a 1:1 mix of halocarbon oil 27 and 700 (Sigma-Aldrich). Embryos were injected using a Transferman NK2 micromanipulator (Eppendorf, Hamburg, Germany), and a PV820 microinjector (WPI, Sarasota, FL) attached to the spinning disk confocal microscope. Drugs (Y-27632, Tocris Bioscience, Bristol, UK); (Cytochalasin D, EMD Millipore, Darmstadt, Germany) were injected into the perivitelline space, where they are predicted to be diluted 50-fold (Foe and Alberts, 1983 (link)). Y-27632 was injected at 100 mM in water; control embryos were injected with water. Cytochalasin D was injected at 5 mM in 50% DMSO; control embryos were injected with 50% DMSO. Embryos were imaged immediately after injection for at least 10 min.
Halocarbon oil 27 and 700
Manufactured by Merck Group
Halocarbon oil 27 and 700 are specialty fluids produced by Merck Group. These oils are designed for use in laboratory equipment and instrumentation. Halocarbon oil 27 and 700 provide thermal and chemical stability for various applications. Their core function is to serve as heat transfer and insulating fluids in controlled environments.
Automatically generated - may contain errors
Lab products found in correlation
Oil immersion lens, by Olympus (3 mentions)
Ixon ultra 897 camera, by Oxford Instruments (3 mentions)
Revolution xd, by Oxford Instruments (2 mentions)
Nk2 micromanipulator, by Eppendorf (1 mentions)
Y 27632, by Bio-Techne (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Ultra 897, by Oxford Instruments (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
5 protocols using halocarbon oil 27 and 700
1
Microinjection of Embryos with Drugs
2
Visualizing Embryonic Development Using Confocal Microscopy
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Stage-7 embryos were dechorionated in 50% bleach for 90 s, rinsed, glued ventrolateral side down to a glass coverslip using heptane glue, and mounted in a 1:1 mix of halocarbon oil 27 and 700 (Sigma-Aldrich, St. Louis, MO). Embryos were imaged using a Revolution XD spinning disk confocal microscope equipped with an iXon Ultra 897 camera (Andor, Belfast, UK) and a 1.5x coupling lens. For experiments using laser ablation, a 60x oil immersion lens (Olympus, Shinjuku, Japan; NA 1.35) was used; for all other experiments, a 40x oil immersion lens (Olympus, NA 1.35) was used. Sixteen-bit Z-stacks were acquired at 0.3-µm steps every 3–10 s (8–10 slices per stack).
3
Embryo Imaging via Spinning Disk Confocal Microscopy
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Stage 14-15 embryos were dechorionated in 50% bleach for 2 min, rinsed, glued ventral-lateral side down to a glass coverslip using heptane glue, and mounted in a 1:1 mix of halocarbon oil 27 and 700 (Sigma-Aldrich, St. Louis, MO) (Scepanovic et al., 2021) . Embryos were imaged at room temperature using a Revolution XD spinning disk confocal microscope equipped with an iXon Ultra 897 camera (Andor, Belfast, UK), a 60x oil immersion lens (Olympus, NA 1.35) and Metamorph software (Molecular Devices). Sixteen-bit Z-stacks were acquired at 0.5 μm steps. Maximum intensity projections were used for markers that localized strongly to the apical surface of cells.
For markers that localized throughout the cells (e.g. Rap1:GFP, Rap1 activity biosensor, or Ephexin:YFP), LocalZProjector (Herbert et al., 2021) was used to identify the apical surface of the embryos and create a maximum intensity projection of the 3 planes surrounding the apical plane.
For markers that localized throughout the cells (e.g. Rap1:GFP, Rap1 activity biosensor, or Ephexin:YFP), LocalZProjector (Herbert et al., 2021) was used to identify the apical surface of the embryos and create a maximum intensity projection of the 3 planes surrounding the apical plane.
4
Imaging Embryonic Cardiac and Epidermal Dynamics
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Stage 14 embryos were dechorionated in 50% bleach for 2 minutes, rinsed, glued dorsal side down to a glass coverslip using heptane glue, and mounted in a 1:1 mix of halocarbon oil 27 and 700 (Sigma-Aldrich, St. Louis, MO) (Scepanovic et al., 2021) . Embryos were imaged using a Revolution XD spinning disk confocal microscope equipped with an iXon Ultra 897 camera (Andor, Belfast, UK), and using a 60x oil immersion lens (Olympus, Shinjuku, Japan; NA 1.35). Sixteen-bit Z-stacks were acquired at 0.75 µm steps every 15-30 s (21-27 slices per stack). Maximum intensity projections were used for analysis in the heart. In the epidermis, Z-stacks were projected using a local Z projector to correct for the curvature of the embryo (Herbert et al., 2021) . To image the dynamics along the entire length of the heart, 3 overlapping positions along the embryo were imaged as above and maximum intensity projections were stitched using a feathering blending algorithm, where the weighting coefficients at the stitching seam between two input images were calculated based on the distances of each input from the seam (Uyttendaele et al., 2001) .
5
Imaging Embryonic Cardiac and Epidermal Dynamics
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Stage 14 embryos were dechorionated in 50% bleach for 2 minutes, rinsed, glued dorsal side down to a glass coverslip using heptane glue, and mounted in a 1:1 mix of halocarbon oil 27 and 700 (Sigma-Aldrich, St. Louis, MO) (Scepanovic et al., 2021) . Embryos were imaged using a Revolution XD spinning disk confocal microscope equipped with an iXon Ultra 897 camera (Andor, Belfast, UK), and using a 60x oil immersion lens (Olympus, Shinjuku, Japan; NA 1.35). Sixteen-bit Z-stacks were acquired at 0.75 µm steps every 15-30 s (21-27 slices per stack). Maximum intensity projections were used for analysis in the heart. In the epidermis, Z-stacks were projected using a local Z projector to correct for the curvature of the embryo (Herbert et al., 2021) . To image the dynamics along the entire length of the heart, 3 overlapping positions along the embryo were imaged as above and maximum intensity projections were stitched using a feathering blending algorithm, where the weighting coefficients at the stitching seam between two input images were calculated based on the distances of each input from the seam (Uyttendaele et al., 2001) .
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