The overall goal of this procedure is to describe an in vivo technique to image subcellular dynamics in sea elegance embryos exposed to anoxia using a gas flow through setup on a high powered microscope. This is accomplished by first preparing a sample of young adult transgenic animals to generate a suitable population of young embryos for imaging. Next, prepare thin agros pad slides for use in the micro incubation chamber.
Then the sea elegance are properly anesthetized and covered with halo carbon oil to avoid desiccation when exposed to the gas flow. Finally, the round cover glass with the anesthetized animals is placed into the micro incubation chamber. The chamber is then placed on the microscope stage and filled with nitrogen gas to produce an anoxic environment while imaging ultimately the use of a gas flow through micro incubation chamber.
In conjunction with in vivo fluorescence microscopy allows high resolution detailed videos and images of cell cycle arrest in response to anoxia. The main advantage of this technique over existing methods like indirect immunofluorescence, is that subcellular changes can be documented in vivo while animals are exposed to specific conditions. Though this method can provide insight into anoxia induced suspended animation in elegans embryos.
It can also be applied to other systems such as yeast, cell culture, or other small invertebrate or vertebrate embryos. Additional conditions such as hyperoxia temperature, stress, or treatment with small molecules can also be applied. Demonstrating this procedure is Anastacia Garcia and Mary Latt two graduate students from my laboratory Begin with an appropriate transgenic C Elgan strain of interest in this study, the TH 32 transgenic strain will be used to visualize chromosomes and centrosomes as markers for cell division.
Generate a synchronized population by disintegrating gravity adults in hypochlorite solution and use remaining embryos picking gravity adults onto a seeded plate and egg, laying one to two hours, or picking L four larvae from a mixed population onto a new seeded plate. Grow nematodes at 20 degrees Celsius to GR young adulthood, which will take approximately 96 hours from hatching, 72 hours from L one larvae, or 24 hours post L four molt. This methodology provides a means to generate a population of young adults that contain an abundant number of young embryos, which contain large blasphemers and nuclei that are optimal for imaging subcellular and sub nucle changes.
To set up a humid chamber to hold prepared slides, place a damp paper towel inside of a large Petri dish or bin covered with a lid of sufficient size. Prepare molten 2%aros in deionized water by heating the mixture in glassware via a microwave or water bath. Next place, one to three drops of warm aros on a clean glass microscope Slide immediately place a second slide inverted perpendicularly on top of the aros drops, and press slightly so that the resulting pad will be thin and free of air bubbles.
After the aros has cooled slowly take apart the two microscope slides, leaving the aros pad intact on one of the slides. It is important to ensure the aros pad is thin enough to not interfere with the ability to focus the microscope.Later. Use a clean razor blade to shape the Aros pad into a small square store.
Slides in the prepared humid chamber until ready to use. Using the same clean razor blade, carefully take the edge of the aros pad and transfer it from the slide onto a round 25 millimeter micro cover glass. Avoiding the accumulation of air bubbles underneath the round cover, glass should fit appropriately in the micro incubator.
Add a drop of anesthetic onto the aros pad. Pick five to 10 worms and place them into the drop of anesthetic. Allow one to three minutes for worms to cease movement.
Using a pipette tip, add a drop of hallow carbon oil on top of the worms to prevent the worms from dehydration as gas is flowed through the chamber. Next, connect one end of the flexible plastic tubing to the chamber. Separate the two halves of the closed perfusion micro incubator and carefully place the cover glass upright onto the bottom ring.
Close the two sides of the chamber tightly together. Then connect the chamber via the flexible plastic tubing to the nitrogen gas tank and place it on the microscope stage. Check for any leaks by ensuring secure attachment of the tubing and port plugs along the side of the chamber to image embryos.
First, locate animals at lower magnification. Then move to the appropriate higher magnification. To image embryonic blasphemers in adults, use the 488 nanometer laser to locate the GFP fusion protein in the cells of interest.
To visualize a specific stage of cell cycle arrest, for example, late prophase, identify a blasphemer at a prior stage of cell division such as late interphase or early prophase. Perfuse the chamber with nitrogen gas and continue to monitor the blasphemers as the nitrogen fills the chamber, adjusting the focal plane as necessary. Conduct time lapse imaging to capture the desired phenomenon of interest in this case.
Prophase arrest and docking of chromosomes to the inner nuclear membrane. Take images once every 10 seconds for up to 30 minutes. Typically, anoxia induced prophase arrest occurs within 20 to 30 minutes of beginning nitrogen gas flow.
To document recovery from the anoxia induced arrested state, turn off the gas and allow the chamber to return to normoxia while recording a time-lapse series. Resumption of cell cycle and undoing of chromosomes typically occurs within five to 20 minutes. Use erris image J or Photoshop to process images and videos and import them into QuickTime for display.
Within the embryo exposed to anoxia, the chromosomes of prophase. Blasphemers will condense and align with the inner nuclear periphery. A phenomenon referred to as chromosome docking upon reoxygenation cell cycle progression will resume and chromosomes of prophase.
Blasphemy will move from the nuclear periphery to the equatorial plate indicating progression to metaphase. Once mastered, this technique can be performed in three to four hours using appropriately staged animals. While attempting this procedure, it's important to remember to use only healthy young adult animals to set aside enough time to complete the experiment and to be patient when learning to choose appropriately staged blasphemers.
After watching this video, you should have a good understanding of how to capture anoxia induced subcellular events in vivo using GaN embryos and a gas flow through chamber on high powered microscope.
