The arrowhead points to the boundary between Diencephalon and Mesencephalon. not in ventral Diencephalon (di). (B) A sagittal section shows that is expressed in the Mesencephalon (mes), Diencephalon (di), but not in the Metencephalon (met) and Pons. The arrowhead points to the Rabbit Polyclonal to AQP12 boundary between Diencephalon and Mesencephalon. Dashed lines demarcate the boundary between the midbrain and hindbrain. is: Isthmus. The mounted sections can be stored at 4C. 4.?Notes: The 24-well plates are for the convenient storage of individual small sample, such as the brains of E12.5 embryos or younger. Vials of appropriate size should be used for older/bigger brain samples. Distilled, de-ionized water (ddH2O) used in this protocol is ultra-purified water with a resistance of 18.2 M. 16% PFA should be prepared in a fume hood because PFA is highly volatile and irritant. It is critical to use Superfrost Plus slides as they contain a special coating to prevent the sections from falling off the slides during the incubation and washing steps. Make fresh blocking buffer for each experiment. Do not store for more than a week. Dabco is irritant. Avoid contact with skin and eyes. Avoid inhalation of vapor or mist. Ethidium is highly carcinogenic. Avoid direct contact and dispose Ethidium-containing waste properly. DEPC is toxic. Avoid direct contact. All DEPC-treated solutions need to be autoclaved to degrade DEPC, which can react with RNA. When thawing the DIG-labeling mix, avoid prolonged incubation at 37C to reduce the chance of NTP degradation. Keep acetic Amrubicin anhydride from moisture (keep the container tightly closed all the time) and add acetic anhydride immediately before use. Do not re-freeze proK for RNA in situ hybridization. The activity of proK should be tested every time a new batch is introduced. To make 50% dextran sulfate, mix 10 ml dextran sulfate and 10 ml ddH2O in a 50 ml tube by inverting, shaking, vortexing and heating at 60C. When dextran sulfate starts to get into water, the total volume will decrease. Add more ddH2O to keep the final volume at 20 ml. Sterile RNase buffer can be stored at room temperature. Take caution to avoid contaminating the bench and other lab materials with RNase A. NTMT should be freshly prepared. At this step, we usually remove the extra-embryonic membranes or the tips of the tails to genotype the embryos. The tissue should float on the surface of the 30% sucrose initially, and sink to the bottom of the well after sucrose has fully infiltrated the tissue. Make sure the tissue is completely immersed in the sucrose solution, as the morphology of tissues staying at the air/liquid interface can be distorted by surface tension. The time the embryos immersed in OCT depends on the size Amrubicin and density of the tissue. For large and/or dense tissue, longer time is needed. Do not freeze samples by directly placing the embedding mold in liquid nitrogen. If not cutting sections right away, wrap the embedding mold with parafilm and keep it at ?80C for up to one week. See manufacturers manual for how to cut cryosections. Cryosections can be stored for months at ?80C without noticeable Amrubicin degradation of proteins and mRNAs. Dried sections do not fall off the slides in subsequent experiments. In addition, drying creates holes in the subcellular structure, permeabilizing the cells for Amrubicin further experiments. Take caution not to let the sections dry completely. If necessary, process slides one at a time. This applies to all steps that involve taking slides out of the solution. The antibody solution stays on the top of slides only if Amrubicin the edges and the bottom of the slides are dry. Therefore, add the antibody solution to the center of the slide and avoid moving the chamber once the antibody is added. Place a sign on the chamber to warn others not to move it during incubation. Covering the humidified slide incubation chamber with foil or perform the incubation in a cabinet. Cut the pipet tip to make a large orifice because Dabco is sticky. To avoid bubbles, lower the coverslip slowly. The fluorescent signal decreases over time, so try to observe the fluorescence as early as you can. In.