Donor red blood cells are first collected from transgenic globin GFP fish by puncturing the heart of the donor animal and aspirating the red blood cells. Once the cells are prepared for injection recipient fish are injected retroorbital and later imaged using standard whole animal fluorescence microscopy for successful injection of fluorescently labeled cells. Similarly, a colored dye dextrin conjugated to Texas red is prepared and injected retroorbital and the injected animal is imaged for successful injection.
Hi, I'm Emily Atch from the Laboratory of Leonard Zan in the Department of Hematology and Oncology at Children's Hospital Boston. Today we'll show you a procedure for Retroorbital injection in adult zebrafish. We use this procedure in our laboratory to study hematopoietic stem cell transplantation.
So let's get started. We will be demonstrating the retroorbital injections of transgenic globin GFP cells and a red fluorescent dye dextrin conjugated to Texas red into adult Casper fish, A transparent breed of zebra fish to prepare transgenic lobin. GFP cells bleed adult donor fish by using a 10 microliter pipette tip coated in heparin to puncture the fish behind the gills, aspirate red blood cells and dispense them into a cell buffer.
Next, filter the cell suspension over 40 micrometer mesh. After determining the concentration of the cells using a hemo cytometer as previously described by LeBlanc etal spend on the cells at 1500 RPM for five minutes. Reus suspend the cells in cell buffer at a desired concentration such that the final injection volume is not more than five microliters.
In this demonstration, we will inject 1.5 to 2 million cells per recipient to prepare the red fluorescent dye, dissolve it in DPBS for a final injection concentration of 10 to 12 milligrams per milliliter. We will be injecting four microliters per fish since the injection procedure is the same. Regardless of the injected material, we will only demonstrate injection of the red fluorescent dye.
Prior to injecting the fish, wash the Hamilton syringe three to four times using 70%ethanol. Rinse three to four times with 0.9 XDPS. After anesthetizing the fish in Trica, place the fish dorsal side up and facing right on a damp sponge.
Hold the loaded Hamilton syringe with your right hand and with your index finger on the plunger with your left hand gently stabilize the body of the fish. Position the needle with the bevel facing up, such that if the fish's eye were a clock, the needle would be pointing at the seven o'clock position. And at a 45 degree angle to the fish, gently insert the needle one to two millimeters into the seven o'clock position and slowly depress the plunger.
Allow the fish to recover in fresh E three water. Keep fish off flow for one week with daily water changes to avoid infection, we keep fish in ICU water for this period. When retroorbital injections are performed correctly, it is possible to visualize injected material that has been labeled.
For example, transgenic globin GFP red blood cells can be seen under a fluorescent dissection microscope circulating in the vasculature of the tail of a recipient Adult Casper fish three days after injection, likewise successful injection of a 70 kilodalton dextrin conjugated to Texas red immediately produces fluorescent vasculature. In the transparent adult Casper fish, which is readily visualized by standard whole animal fluorescence microscopy, fluorescent hole kidney marrow cells from transgenic beta actin GFP donor fish can also be injected retro orally into a radiated Casper recipient fish. These cells will eventually home to the recipient marrow as shown here, three days following injection, four weeks later, these green donor cells may go on to repopulate the recipient kidney.
We've just shown you how to perform retroorbital injections in adult zebrafish. When doing this procedure, it's important to remember to inject with your needle at a 45 degree angle to the plane of the fish. So that's it.
Thanks for watching and good luck with your experiments.
