The overall goal of this procedure is to perform immuno staining on dissected zebrafish embryonic hearts to reveal cellular or subcellular structures in high resolution. This is accomplished by first preparing slides in a humidified chamber for immuno staining. Next, the heart is dissected from an anesthetized zebrafish embryo, followed by immuno staining on the embryonic heart with a specific antibody.
Then images of fish hearts are documented with a microscope equipped with an aome. Finally, cellular or subcellular structures in a zebrafish embryonic heart are revealed using immunofluorescence microscopy Due to much increased penetration. The male advantage of this technique over existing methods like home mount immuno staining, is to be able to obtain high quality images of standard zebrafish heart To prepare a humidified chamber and to protect samples from light, use aluminum foil to wrap the chamber and the cover of an empty tip box.
Prevent the samples from drying by placing a stack of wet paper towels in the bottom of the chamber, and place a small microplate on top of the paper towels as a rack for slides. Using an image pen to make wells draw either circles or lines with dimensions of about five by five millimeters on the surface of a microscope slide. Let the slides dry.
Put the slides in the humidified chamber and add 50 microliters of formaldehyde to each well. To dissect the embryonic heart, anesthetize the fish embryo in E three egg water containing 0.4%trica until the fish stops all body movement, but still maintains normal heart beating. About one minute.
Transfer the embryos to a concave. Well place a concave microscope slide on the stage of a dissection microscope using either dark field or DIC like polarized light. Adjust the light of the microscope to clearly reveal the heart.
Next, clean two insulin syringes with 75%ethanol. Then dry, increase the magnification of the microscope to focus on the heart. Next, physically fix an embryo by inserting one needle tip into the yolk, so mite boundary close to the head.
Then insert another needle close to the heart region, and quickly pull out the heart. If the heart is not totally separated from the embryo, cut off the remaining connected tissue between the heart and the yolk. Focus on the heart at a lower magnification.
With a 10 microliter pipette man set to a one microliter volume. Apply the tip of the pipette man to either draw up or touch the heart sample so that the isolated heart will be either within or attached to the surface of the pipette tip. Dip the pipette tip into the formaldehyde solution on prepared slides and depress the pipette piston to release the heart into the solution.
With the heart's in formaldehyde on the depression slides, close the chamber and place it on a shaker with gentle rocking for 20 minutes at room temperature, wipe off the water on the back of the slides and place the slides on the stage of a dissection microscope for buffer exchanging. Use a 10 microliter pipette man to carefully aspirate the formaldehyde from the slide while leaving the hearts undisturbed. Add 50 microliters of PBST to cover the heart samples and incubate in the humidified chamber for five minutes.
Rinse the hearts a second time in PBST. Next, block the heart sample with 10%sheep serum in PBST for one hour at room temperature. Then incubate the heart samples with one to 200 dilution of beta catenin and F two specific primary antibodies in 10%sheep serum PBST for one hour at room temperature.
Wash the heart samples with PBST three times for five minutes each at room temperature. Incubate the samples with a one to 1000 dilution of a Alexa tag, anti muse and or anti rabbit secondary antibodies in PBST for 30 minutes. Wash the samples again three times with PBST to image the samples.
Remove the PBST and add one microliter of mounting medium to each. Well rock the chamber for several minutes until the solution becomes clear. Image the fish heart using a Zes Axio plan two microscope equipped with an aome In this image, the nuclei and cell membranes of zebra fish cardiomyocytes from a zebra fish embryo at 52 hours post fertilization are identified with antibodies against MEF two C and betaine respectively.
By adjusting the stage of the compound microscope, images of the heart samples can be adjusted to the same orientation, which allows the observation of the outer curvature and inner curvature of the heart. The total cardiomyocyte number cardiomyocyte cell size and circularity can then be measured Here, the sarcomeric structure of a dissected embryonic heart is shown using the myosin antibody F 59. The Myo fial network in a whole embryonic heart ventricle is clearly visible at lower magnification while the striated band of thick filaments is seen at higher magnification.
After watching this video, you should have a good understanding of how to perform immunostaining dissected ebro zebrafish heart, especially, how to dissect heart from zebrafish April, and how to handle the heart during immunostaining process.Process.
