For embryos up to 62 hpf, acquire Z-stacks of the endocardium using 561 and 488 nanometers for red and green fluorescent signals, respectively. Return embryos to the dark at 28.5 degrees Celsius to allow them to continue to develop normally.Īt the desired stage, stop the heart and re-embed the embryos, as demonstrated earlier in this video, with the important difference that the embryos must be treated with BDM in separate, marked dishes to keep track of them. Now that the embryos have been removed from the BDM containing medium, their hearts should start beating again in about five minutes. Transfer the embryo to a well in a six-well plate containing embryo medium with PTU.ĭuring this step, make sure to keep note of which embryo goes into which well, as this is essential to correlate the position of photoconverted cells at later developmental stages. To wash away the BDM containing medium, eject the embryo from the glass pipette into an 85-millimeter Petri dish containing embryo medium with PTU. After photoconversion, use a glass pipette to press down slightly just above the head of the embryo to break the LMP agarose. Return to the TCS SP8 mode on the software, and using the 561 and 488-nanometer lasers sequentially, in Bidirectional mode, acquire a Z-stack. Should insufficient Kaede be photoconverted in the ROI, scan the 405-nanometer laser over the area three times again. Then, using the 405-nanometer diode laser at 25%laser power, photoconvert the cells, scanning over the ROI three times.

While focusing on the plane of interest, select a region of interest, or ROI. Then, select approximately 1%laser power for both the 488 and the 561-nanometer lasers. Next, on the microscope acquisition software, enter FRAP mode. Then, use the 488-nanometer laser to visualize the endocardium. Using the 561-nanometer laser, check for red Kaede fluorescence. To carry out photoconversion, on an upright confocal microscope equipped with a 405, 488, and 561-nanometer laser sources, use transmitted white light and an objective lens to locate the embryo's heart. The embryos are now ready to be photoconverted. Wait approximately five minutes for the LMP agarose to set, then add mounting medium to the dish. Press down on the walls of the indented square to allow excess agarose to overflow to the side areas of the mounting dish. Then, readjust the embryo positions, tilting them to the left or right as necessary. When the embryos are roughly in place, remove the medium, and embed them in approximately 200 microliters of 0.7%LMP agarose containing tricaine and BDM. Then, when the hearts have stopped beating, transfer the embryos to the mounting dish and use forceps to arrange them in the wells so that they lie at a 25-degree angle, tails pointing toward the deeper part of the well, with the yolk facing up. Transfer the embryos to a separate dish containing two milliliters of mounting medium. Then, transfer the tube to a 38-degree Celsius heat block to keep the LMP agarose in its liquid state. Then, add 25 microliters of eight milligram per milliliter tricaine and 40 microliters of one molar BDM solutions to the tube. Pipette the LMP agarose up and down to cool it slightly. In the meantime, melt a one-milliliter aliquot of 0.7%low melting point, or LMP, agarose by placing the tube in a 70-degree Celsius heat block for about five minutes. Next, add two milliliters of previously prepared mounting medium to the mounting dish, and allow 15 to 30 minutes for the solution to diffuse into the agarose. Then, remove the plastic mold from the agarose. Place the dish at four degrees Celsius until the agarose hardens. Then, position the plastic mold in the liquid agarose, taking care to avoid trapping air between the mold and the agarose.

Demonstrating the procedure will be Renee, a postdoc in my lab.Īfter creating a mold according to the text protocol, pipette about 1.5 milliliters of melted 1%agarose into a 35-millimeter plastic mounting dish. The main advantage of this technique is that it bypasses some of these difficulties. It makes it difficult to follow cells via traditional time-lapse microscopy. Tracking cells during AVC and valve formation is challenging because of the rapid beat of the heart. This method can help to address key question in the field of cardiac development, such as to understand how endocardial cells behave in different types of mutants. The overall goal of this procedure is to follow endocardial cells during atrioventricular canal and heart valve formation.

Photoconvert