In vivo live imaging using two-photon microscopy

Pregnant CX3CR1-GFP mice were anesthetized with isoflurane (Cat#099-06571, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) inhalation. To facilitate the handling of uterine horns, 2 mg per kg ritodrine hydrochloride (Cat#186-02231, FUJIFILM Wako Pure Chemical Corp.), a myometrium relaxant, was intraperitoneally administered. After a midline laparotomy was performed, the distal part of one uterine horn was fully mobilized by cutting the mesometrium and ligating the ovarian vessels (this procedure did not affect uterine circulation or embryos’ survival until birth). Then, an embryo (in the uterine tube) was sandwiched by two holding bars equipped in a box, to be subsequently used for further fixing of the embryo with low-melting temperature agarose (5%). The head of the embryo was positioned to make the dorsolateral part of the cerebral hemisphere closest to the objective lens. A coverslip attached to a water container (for immersing the objective lens) was gently pressed to the uterine wall and was tightly held to the subjacent equipment. Throughout the preparative and imaging steps, the mother mouse was warmed using a heater to maintain body temperature. Pallial walls were scanned by an A1RMP two-photon microscopy (Nikon, Tokyo, Japan) using a 20× (N.A. 1.0) water immersion lens (Fig. 1j–o).