In ovo technique for cell injection in the CPM followed by bead implantation in the BA2 of chicken embryos

Highlights • Our method involves bead implantation followed by quail cell injection and provides useful tools for tracing migratory mesodermal cells in vivo.• The proposed method does not require any commercial kits and can be used for various developmental process.• It does not employ any complicated methods such as genetically engineered permanent cell labeling or multiplicity of fluorescent markers.


Chicken embryo model and egg preparation
Fertilized chicken eggs (Gallus domesticus) were obtained from a local poultry breeder and stored in the fridge (Fig.1A) at 8À16 C. The eggs were rinsed with 70 % ethanol and incubated (Fig.1B, J. Hemel egg incubator, Verl, Germany) at a temperature of 37 C and at 80 % relative humidity until the stage HH10-11. At this stage, a hole was made in the side of the air chamber using small surgical scissor and 2-3 ml of albumen were withdrawn to lower the embryo using a sterile syringe ( Fig. 3 and Video 1). The upper side of the egg was covered with medical tape. An oval window about 2 cm in length was opened on the same side of the egg.

Materials necessary to make videos
The Olympus teaching stereo microscope has a dual head of binocular; one is for objective lens (main head) and the other one is for digital camera (secondary head) (Fig. 2B). We used the secondary head to attach the camera of Samsung Galaxy S8 ( Fig. 2A) to the eyepiece. All videos were edited using MovieZilla Movie Maker Software.

In ovo quail-chicken transplants
Quail cells were grown in DMEM (DMEM; Invitrogen, USA) supplemented with 10 % fetal calf serum (Invitrogen) in a 37 C humidified atmosphere of 5 % CO 2 in air. Cells were seeded on the day of   the injection at a density of 3 Â 10 5 cells per T25 flask. Cells were harvested, centrifuged in DMEM onto microcentrifuge tube. The eggs were windowed and prepared for cell injection following the procedures previously described above. India ink diluted 1:10 in Locke's solution containing penicillin G (Penicillin G sodium salt, PENNA, Sigma), was injected into the yolk under the blastoderm to allow visualization of the embryo. The extra membrane overlaying the embryo (vitelline membrane) was partially removed with a tungsten needle. The cell injection technique continuously used chick embryos as hosts for quail cells. The cells were injected in the cranial paraxial mesoderm of HH10-11 chicken hosts using a capillary glass needle (Video 2 and Fig. 5A). The operated eggs were sealed with medical tape and re-incubated for 24 h (HH15-16).

Preparation of SDF-1 beads
The AG beads (AG 1-X2 resin, 143-1255, Bio-Rad) were placed on the top of the microcentrifuge cap and soaked in a 1 mg/ml SDF-1 (300-28A; PeproTech). Control beads were soaked in PBS. The caps with beads were afterwards placed into small Petri dish surrounded by Cellulose compress (900-0853, Henry Schein). Few drops of Locke's solution were adsorbed onto Cellulose compress, to create a humid environment which will avoid the dryness of the beads. The dishes were sealed with parafilm and kept at 4 C overnight ( Fig. 4 and Video 3).

In ovo bead implantation
When the injected embryos reached stage HH15-16, the amnion membrane overlaying the embryos was carefully removed with a tungsten needle. An incision in the BA2 tissue was created with a fine tungsten needle. Using fine forceps, the beads were inserted into the incision. PBS beads were implanted as a control. The operated eggs were sealed with medical tape and re-incubated until the stage HH19-22 (Video 4 and Fig. 5B). Finally, the embryos were fixed in 4 % PFA in PBS and processed for whole mount immunostaining.

Whole mount immunostaining
The whole mount immunostaining method presented here is based on the method by Kodo K. et al.[1]. Embryos ranging from stage HH19 to HH22 were isolated and fixed overnight in PFA 4 % in PBS at 4 C. The fixed embryos were dehydrated in a series of different graded of methanol in PBS at room temperature and were subsequently stored at À20 C. Embryos were then incubated in 6 % H 2 O 2 in methanol for 6 h at room temperature to block the endogenous peroxidase activity. The embryos were then rehydrated and blocked in blocking solution (PBST, 2 % non-fat milk, 1.0 % Triton X-100) for 1À2 h at room temperature. To detect quail cells, embryos were incubated with the primary antibody (mouse anti-quail QCPN monoclonal antibody (from Developmental Studies Hybridoma Bank, the university of Iowa)), which is specific to quail cells (Fig. 5C, D), in blocking solution (diluted 1:50) for 2 days at 4 C. After five times washes (1 h for each wash) in blocking solution at 4 C, the embryos were incubated with HRP (horseradish peroxidase) conjugated with the secondary antibody (goat anti-mouse (polyclonal, 1:200, Jackson ImmunoResearch Lab, USA)) in blocking solution at 4 C overnight. The following day, embryos were washed extensively 5 times (1 h for each wash) in blocking solution at 4 C. The staining was performed by incubating embryos in the staining solution (250 ml DAB (0.16 mg/ml), 1750 ml PBS and 2 ml H 2 O 2 30 %) for 15 min at room temperature for color reaction to develop. After removing the staining solution, the embryos were rinsed with PBS for 10 min at room temperature followed by the fixation overnight in 4 % PFA in PBS at 4 C. Images were taken using a stereo microscope (M165 FC, Leica, Germany) equipped with a digital camera (DFC420 C, Leica, Germany) (Fig. 5).

Declaration of Competing Interest
The authors declare that there are no conflicts of interest.