Mice are maintained on a 12-h light cycle with light being turned on at 07:00. Animals are anesthetized with isoflurane (Abbott Laboratories) and either killed by decapitation (for brain preparations) or through cervical dislocation (for embryo collection).
Tissues are processed as follows:
E10.5 embryos: Embryos are fixed in 4% paraformaldehyde at 4°C over night. Two alternative methods are used for embedding:
Method 1: Embryos are cryo-protected with sucrose and embedded in gelatin and frozen in special copper chambers (see below).
Method 2: Embryos are embedded in paraffin using standard methods of paraffin embedding.
E14.5 embryos, heads of E15.5 embryos: Following a brief wash in cold phosphate-buffered saline, tissue is transferred to ice-cold O.C.T 4583 (Tissue-Tek, Sakura). Tissues are transferred to a freezing chamber (Image 3) within 5 min after dissection. A freezing chamber consists of a square copper base and transparent Plexiglass side walls. The chamber is placed on a specially designed, movable stage fitted to a stereomicroscope. Through translation and rotation of this stage, the edges of the chamber can be aligned parallel to a grid placed into one of the eyepiece of the stereomicroscope. The chamber is filled with O.C.T. and placed for 40 sec onto an aluminum block submerged by 2/3 in a dry ice/methylpentane mixture (maintained at -65°C). The chamber is then removed and the tissue is submerged in O.C.T. and oriented appropriately with the aid of a blunt dissecting needle. Orientation is achieved if the dorsal midline of the embryo is parallel to the gridlines of the eyepiece and the sagittal midplane of the brain is parallel to the side walls as judged by viewing the specimen frontally through the transparent side walls. After orientation, usually within 1 min, the chamber containing the specimen is returned to the cooling device where it continues to freeze in a uniform fashion with the plane of freezing moving upward at a rate of about 3 mm per minute. Following freezing, the chamber is disassembled. O.C.T. blocks containing tissue are placed into plastic bags and are stored at –80°C for several months. Gelatin embedding of E10.5 embryos is carried out in an analogous manner. Embryos are oriented with their mid-plane parallel to the copper plate.
P7 and P56 brains:The dissected brain is placed into a dish filled with ice-cold O.C.T. and shortly thereafter the tissue is placed and frozen in the freezing chamber as described above. Orientation: midline of the brain is set parallel to the grid of the eyepiece and equivalent structures of the brain are equidistant to the copper plate, as judged by viewing the brain frontally through the transparent side walls.