The overall goal of this procedure is to efficiently isolate skeletal muscle fibers from embryonic and larval zebra fish. This is accomplished by first preparing poly L lysine coated cover slips next one to four days post fertilization, zebrafish embryos are dissociated. Then myo fibers isolated from the dissociated embryos are plated on the polylysine cover slips.
Finally, the myo fibers are fixed, stained and imaged for analysis of muscle proteins. Ultimately, results can be obtained that show subcellular localization of muscle proteins through immunofluorescence microscopy. The main advantage of this technique over existing methods like whole embryo analysis, is that it allows for the study of single muscle fiber morphology and physiology in greater detail.
This method can help answer key questions in the muscle field, such as those that pertain to subcellular protein localization, and it can be combined with live cell calcium imaging. The implications of this technique extend toward validation of zebrafish models of muscle disease because it can be used to visualize pathognomonic structural abnormalities associated with human disease. It can also be used to help identify new aspects of muscle disease.
Pathogenesis Through this method can provide insight into muscle structure and function. It can also be applied to other studies such as human skeleton muscle diseases. To get ready for the dissociation of zebra fish embryos, prepare covers slips by cutting and placing parfum on the bottom of a 60 millimeter Petri dish.
Then place glass cover slips onto the parfum and pipette 50 to 200 microliters of poly lycine solution onto each cover slip. Incubate the cover slips at 37 degrees Celsius for at least one hour before removing the polylysine solution and washing them twice with 100 microliters of PBS. Allow the cover slips to dry, then store them in a covered dish.
For embryo dissociation. Transfer 10 to 23 day post fertilization zebrafish embryos into a standard 1.5 milliliter centrifuge tube and remove as much excess fish water as possible. Pipette 900 microliters of carbon dioxide independent medium into the tube.
Then add 100 microliters of 3.125 milligrams per milliliter collagenase, two or four solution to begin dissociation. Rotate the embryos on an orbital shaker at room temperature and every 30 minutes use a P 1000 pipe Pepin to iterate the sample to prevent over digestion when no whole embryos are visible, yet solid pieces are present. Stop the reaction by pelleting the samples at 0.8 to 2.3 Gs for three to five minutes.
Remove the supernatant and wash two times with carbon dioxide independent medium. Pass the suspension through a 70 micrometer filter to remove debris at about 50 to 100 microliters of myofiber suspension to each poly L lysine coated cover slip. Allow the myo fibers to settle for an hour at room temperature after fixing and immuno labeling the myo fibers according to the text protocol mount.
The cover slips by first applying one to two drops of Antifa reagent to a slide. Use forceps to carefully pick up a cover slip, invert it, and place it on the drops of anti fade reagent on the microscope slide. Next, lay one to two tissues on a hard solid surface and quickly invert the slide onto the tissues.
Apply light pressure to the corners of the slide to remove excess Antifa reagent and form a tight seal between the slide and cover slip. Allow the slides to dry for five to 10 minutes at room temperature before imaging or stored at four degrees Celsius. Shown here, myofibers immuno fluorescently labeled against the anodyne receptor and alpha actinin reveal the triad and the zand respectively.
We have utilized isolated myop fibers in previous studies to determine subcellular localization of muscle proteins. To study the localization of chimeric expressed transgenes and to define specific myofiber parameters including size. This figure demonstrates normal caffeine induced calcium release in myop fibers expressing the PSKM GCaMP three construct.
The technique as outlined in the text protocol can be used to interrogate the excitation contraction coupling apparatus via live imaging Following this procedure. Other methods like live calcium imaging can be performed in order to answer additional questions like, are there defects in excitation contraction coupling After its development? This technique paved the way for researchers in the field of muscle disease research to explore validation studies of newly identified muscle disease genes and determine novel aspects of muscle disease pathogenesis.
After watching this video, you should have a good understanding of how to isolate the individual mild FBS from zebrafish ems.
