Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

All animal procedures were approved by the CNIC Animal Experimentation Ethics Committee, by the Community of Madrid (Reference PROEX 220/15) and conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.

This protocol includes the use of two males from the fluorescent transgenic mouse line reporting NOTCH activity Tg(CBF:H2BVenus,+)¹⁸. See the Table of Materials for details about materials, animals, and equipment used in this protocol.

1. Tools and holder customization

1. Cut 1 cm long tungsten wire pieces and sharpen them to 0.02-0.05 mm diameter using any of the simple methods available, for example, submerging the tip of the wire in a saturated sodium nitrite solution¹⁹.
2. Prepare elbowed glass capillaries.
   1. Place the midpoint of a standard 1.0 mm glass capillary over a Bunsen lighter for 3-6 s, pulling slowly from both ends until the capillary turns thin and flexible. At that point, break the capillary in half and heat it briefly to produce a 90° bend 2 cm from the tip.
3. Saw a piece of polymethyl methacrylate measuring approximately 7 mm long, 4 mm wide, and 2 mm thick (Figure 3D).
   NOTE: Polymethyl methacrylate sheets can be either obtained from recycled laboratory equipment or ordered new.
4. Hold the methacrylate piece using a bench vise. Next, drill custom-sized holes transversally through the whole piece (Figure 3D).
   NOTE: Typically, E6.5 to E7.5 mouse embryos require using 0.2-0.5 mm diameter drills.
   1. Rotate the drill slowly while applying low but constant pressure. If a drill breaks inside the holder, discard the piece, as the metal cannot be taken out.
5. Use a fine file to smooth the holder's edges and adjust its size. Additionally, create a mild slope on the edges of the holder to facilitate embryo positioning (Figure 3D).
6. Place the finished holder in a dish with distilled water to rinse it.
7. Check the holder under the stereomicroscope to see if the holes contain drilling dust. If dust is present, use tungsten needles to remove it.
8. To sterilize the holder, place it in a conical tube full of distilled water and sonicate it for 20 min with a minimum 20 W power output.

2. Media preparation

1. To heat-inactivate the complement system, leave two 500 μL aliquots of commercial or homemade²⁰ rat serum at 56 °C for 30 min.
   NOTE: For a detailed protocol on how to prepare rat serum, see²¹.
2. In a cell culture hood, prepare two aliquots of the medium for culturing the embryos in the microscope. For each, mix 490 μL of DMEM for live-cell imaging, 500 μL of inactivated rat serum, and 10 μL of penicillin-streptomycin to obtain a final concentration of 50 µg/mL penicillin and 50 µg/mL streptomycin²².
   NOTE: The volume of culture medium depends on the time and number of embryos to be cultured. As a rule of thumb, optimal growth requires a minimum of 200 μL per embryo per day in E7.0 embryos.
3. Using a 2 mL syringe, filter the medium through a 0.22 µm pore size membrane filter into a new tube and place it lid-open in a cell culture incubator at 37 °C and 7% CO₂ for 1 h before embryo culture²³.
4. Prepare the dissection medium by adding to a 500 mL of DMEM supplemented with L-glutamine bottle the following: 50 mL of fetal bovine serum, 10 mL of 25 mM HEPES-NaOH (pH 7.2), and 10 mL of penicillin and streptomycin (50 mg/mL).
5. Next, separate it into 50 mL aliquots, place three aliquots in a 37 °C bath and store the rest at 4 °C for up to 3 months.

3. Embryo dissection

1. Euthanize an E6.75-E7.5 pregnant mouse.
2. Extract the uterus and place it on a dry and clean paper wipe. Then, cut the uterus, inserting the tip of fine scissors from the mesometrial side and sliding the blade along to expose the deciduae and transfer them to the dissection medium. For a detailed protocol, see²⁴.
3. Dissect the embryos, keeping the ectoplacental cone intact (Figure 3A,B). For a detailed dissection method, refer to²⁵.
4. Then, peel off the Reichert's membrane, leaving part of it on the extraembryonic region (Figure 3B).
   NOTE: Fine forceps are essential for this step. Inexperienced users can dissect embryos on 2% agarose gel-coated dishes to avoid bending the forceps' tips against the dish surface (Figure 3C).
5. To keep the dissection medium warm, replace it every 10 min. Moreover, dissect deciduae in groups of 3-4 while keeping the rest in dissection medium placed in a 37 °C bath. Immediately place the dissected embryos in culture medium.
6. Select the embryos with the desired fluorescence features using a fluorescence stereomicroscope and place them in the tube containing culture medium in the cell culture incubator.
7. If the embryos are at E6.5 to E7.0 stages, close the lid and leave the embryos to recover for 2 h before imaging.
   ​NOTE: E6.5 to E7.0 embryos are sensitive. They can also be placed directly under the microscope but preculturing allows for selection of the ones that best recover from dissection, increasing the chance of success.

4. Microscope preparation

1. Turn on all microscope components, including lasers, computer, and software. Turn on the temperature controllers and set to 37 °C 3 h in advance to ensure equilibration.
2. If using an immersion detection objective, clean it using a residue-free disposable wipe. Dip the objective's tip in double-distilled water using a 60 mm dish and leave it until the acquisition starts.
3. Turn on the CO₂ controller and set it to 7%.
4. Place a drop of high-vacuum silicon grease on a 35 mm dish with glass coverslip bottom (14 mm diameter), place the holder on top of the drop, and press gently to immobilize it.
5. Under a stereomicroscope, add culture medium to fill the glass-bottom portion of the dish. While doing so, aim the stream to the holder's holes to prevent formation of air bubbles in them.
6. If the bubbles remain, use the glass capillary prepared in step 1.2, connected to a silicone tube with a mouthpiece, to suck the bubbles out gently.
   ​NOTE: The elbowed end of the capillary will facilitate access through the holes.

5. Embryo mounting

1. Transfer 2-3 embryos that were left to recover in the incubator to the dish with the holder. Leave the rest in the incubator as a backup.
2. Set the embryos in the holes by using a pair of forceps and a tapered tungsten needle. Use the needle to hook the embryo by the ectoplacental cone and insert it into a size-matching hole. To immobilize the embryo, rotate the cone by 90-120° so that the Reichter's membrane adheres to the hole walls (Figure 3C). For an alternative description, see²⁶.
3. Carefully move the dish containing the embryos and the holder to the microscope plate (Figure 3F,G).
   NOTE: If not moved steadily, the embryo can move out of the holder.
4. Lower the objective until a liquid meniscus is formed at the medium-objective interface.
5. Locate the embryo using transmitted light under the microscope binocular and place the embryo in focus.
6. Mount the incubation chamber and seal it around the objective using tape (Figure 3F).
7. Using a Live Capture at the microscope control software, adjust the Laser Output and Gain levels.
   NOTE: For two-photon acquisition of green fluorescent protein (GFP) and Tdtomato reporters, 30% output laser power at 980 nm and 70% gain on non-descanned detectors were used in this protocol (Figure 3G).
8. To avoid evaporation, cover the medium with paraffin oil by dripping it slowly on the objective.
   ​NOTE: This is the last point where additional embryos can be mounted in the holder. Once covered with paraffin oil, one would have to clean the holder and replace the medium to do so. For alternative mounting methods see²⁷.

6. Image acquisition

1. Set up time-lapse recording. Set a 5-10 min time interval with 3-5 µm z-space between stacks. Leave a blank space on top of the z-stack to anticipate embryo growth, a minimum of 50 µm, adding 30 µm for every hour the acquisition will not be supervised.
2. If available, enable the Auto-Save option to allow supervision of the acquisition from a personal computer to allow the adjustment of the z-stack size, if needed, to fit the area of interest within focus.

Figure 3: Live imaging tools and setup. (A) Dissecting mouse embryos with an agarose-coated dish under the stereomicroscope. (B) Diagram of the steps to dissect mouse embryos for live imaging. (C) Embryo positioning in the holder. (D) Embryo holder design and features. (F) Incubator chamber around the immersion objective. (G) Diagram of final setup for time-lapse acquisition. Scale bar = 500 µm (A). Please click here to view a larger version of this figure.