If you work in a fish lab, microinjection is probably one of the first techniques you had to learn. Zebrafish make great models for developmental and neurobiological studies, and scientists frequently utilize microinjection to introduce substances like morpholinos, plasmids, RNA, or protein into early-stage embryos. This technique, while relatively simple, can be frustrating when things start to go wrong. Time is of the essence, and researchers often run into several, small obstacles that delay the experiment, resulting in the embryo developing beyond the desired stage.
Here are some tips I’ve learned after years of practice. They go a long way towards staving off a meltdown!
Preparation, Preparation, Preparation!
You’ve probably run into a wild-eyed intern sprinting across the corridors, desperately trying to set up their experiment before the embryos move from the single-cell to the two-cell stage. In fact, you’ve probably been there yourself—I have! Ultimately, being prepared as much as possible goes a long way towards reducing stress on the day of the experiment. Below are some steps you can take to get organized the day before you microinject.
Create an Injection Kit
Prepare an injection kit to ensure you have all the required equipment within arm’s reach on the day of the injection. You will need a box or tray large enough for a micropipette, a box of microloader tips, forceps, a marker, injection needles, an injection mold (optional), and a few plastic Pasteur pipettes.
Prepare an Injection Mold (Optional)
Some people prefer to place the fertilized eggs on a glass slide or Petri dish, while others line up the embryos in an agarose mold to quickly progress between injections. If you belong to the latter category, I recommend preparing injection molds ahead of time. They are easy to make and come in different types. Once made, they can be stored at 4°C and used multiple times.
Pull Your Needles
Pulling needles ahead of time can save you several minutes on the morning of the injection. Use a micropipette puller to pull glass capillaries into needles. If your lab doesn’t have a protocol for this, spend some time experimenting with different needle shapes and sizes. If you do not have a needle holder, simply place a strip of Blu-Tack or putty on a Petri dish, and gently press the pulled needles into it to hold them in place. Always pull more needles than you will need–this way if you end up breaking a few, you won’t have to waste time running back to the puller halfway through your injections.
Dilute Your Reagents
Regardless of what you’re injecting (morpholinos, plasmids, RNA), there will be a desired concentration for your reagents. Wherever possible, dilute these in advance. Ideally, the only thing you want to do the morning of the experiment is to add temperature-sensitive components, such as RNA or enzymes, to create the final mixture.
Cross Your Fish
Once all your equipment and reagents are prepared, it’s time to cross your fish. In my experience, doing this shortly after the fish have been fed generally ensures that they will lay eggs the following morning. Set up as many tanks as you can, preferably with a single pair in each separated by dividers. Fish not laying is often a source of frustration, so it’s always a good idea to increase the odds of receiving fertilized eggs.
On Injection Day
Now that you’ve planned out what you can leading up to the experiment, it’s time to move on to the day of the experiment. The order of these steps is interchangeable, depending on your level of comfort/expertise with the technique.
Prepare the Reagents to be Injected
If you are injecting temperature-sensitive material, get an icebox, thaw the required aliquots, and mix them with the previously diluted reagents. Add Phenol red if needed (this helps to visualize the injection), label the individual tubes clearly, and give them a quick spin down.
Allow the Fish to Mate
Remove the dividers from one or two tanks, and leave the fish undisturbed for a few minutes. On days when the fish just aren’t “feeling it” and won’t lay, you can try tilting the breeding tanks such that one end has a lower level of water. This might encourage the female to lay since they prefer to spawn in shallow waters.
Set up the Injection Apparatus
Turn on the air source, microinjector, and light source for the microscope. If you use a micromanipulator to hold your needle, position it at an optimum distance from the microscope stage. If you have a steady hand or prefer the freedom of doing injections freehand, you can skip this step.
Collect and Arrange the Eggs
Once the injection gods favor you, you can collect the fertilized eggs and proceed with the experiment. Arrange them either in the mold or dish/slide as per your preference, remove excess water either by blotting with a Kim wipe or with a Pasteur pipette, and place them on the microscope stage.
Break the Needle Tip
This is one of the most crucial steps and can take time to perfect. If you are new to microinjections, I recommend loading the needle while waiting for the fish to lay. Leaving the needle tip open for too long can cause it to clog, so it is essential to optimize this step. Practice if you have to!
Backload the needle, and focus the microscope on the thinnest region near the tip. Bring your forceps to the same focal plane, and gently pinch off the glass to create an opening. This should allow a small droplet to come out when pressure is applied but leave the needle sharp enough to pierce the chorion. Adjust the injection pressure and step size accordingly. Remember that less is more. You only want to transfer a few 100 pL to 1 nL per embryo. Once you complete this step, you’re all set to race against time to inject before the cells divide! [Tip: inject into the cell rather than the yolk (Figure 1). You might have to gently rotate the egg with the tip of your needle until you can see the cell.]
While mastering microinjection requires practice and skill, following the above-mentioned tips can increase your chance of success. To summarise: set up as much as you can beforehand, practice as much as you need to, and optimize your process for smooth sailing. Leave a comment below if you have any other suggestions for a successful microinjection. May the fish fertility odds be ever in your favor!