Instruments, Westbury, NY, USA).
and 35-mm Falcon; Becton Dickenson, Bedford, MA, USA).
Technologies, Gaithersburg, MD, USA and Sigma, St. Louis, MO, USA).
Vancouver, BC, Canada and featherblades; Ted Pella, Redding, CA, USA).
ROBOZ Surgical, Rockville, MD, USA).
spatulas, large and small scissors; ROBOZ and Fine Science Tools).
(Millipore, Bedford, MA, USA). Culture Medium To prepare 200 mL, mix together these reagents from Life Technologies, except BME which is from Sigma:
Cover with aluminum foil, medium is light sensitive.
1. Postnatal 5 to 7-day-old rats or 5 to 10-day-old mice are washed with 70% ethanol and decapitated. Their brains are quickly and aseptically removed under a hood. Lateral cuts are made in the skull starting at the foramen magnum and ending at the olfactory lobes. The skull is gently lifted up from the rear exposing the brain. Cuts are then made between olfactory lobes and the frontal cortex and rostral to the cerebellum. The rostral part of the brain is gently lifted to expose the optic nerves, which are then cut prior to removing the brain from the skull. Finally the brain is removed and placed into cold Gey's solution in a 60-mm petri dish containing 0.5% glucose. 2. Carefully remove blood vessels and meninges around hypothalamus and gently straighten out residual optic nerves. These serve as helpful landmarks since the suprachiasmatic nucleus (SCN) is found at the base of optic chiasm. 3. Block out the hypothalamus using a razor blade in a blade holder. Cut away all cortex on both sides lateral to the hypothalamus (from a ventral view) and make a clean horizontal cut 2 to 3 mm above the third ventricle (coronal view). 4. Place the blocked hypothalamus on the tissue chopper so that the hypothalamus is ventral side down on the chopper disk and the optic nerves are abutting the chopper blade. Set the thickness of the slices at 350 to 400 μm for rats and 300 μm for mice. Note: Try to avoid excess fluid on chopper disk as this will cause the tissue to be picked up by the blade. 5. Cut the sections as a group, trying to keep them in order, and lift the group of sections onto a spatula and place them into a cold drop of Gey’s solution plus glucose in a 100-mm petri dish. Carefully separate the slices using a small spatula and forceps. Note: It is critical for the survival of the tissue to be extremely gentle in the transferring and manipulating of the slices at this stage and afterwards. 6. Select the sections of interest and trim them free of extraneous tissue using a razor blade scalpel, place them in fresh drops of the Gey's/glucose solution, and allow them to sit at 4°C in a refrigerator (1–2 h). 7. Put 1.1 mL of culture media into each 35-mm petri dish. The 35-mm petri dishes are placed inside of a 150-mm petri dish acting as a container to minimalize their handling. 8. Place tissue slices to be cultured onto Millicell-CM filters by using two small spatulas touching each other and by using the capillary forces between them to transfer the slices. Make sure the tissue rests flat on the filters. Note: Too much fluid on the filter will prevent tissue from adhering to the filter. If this happens, remove the excess fluid or replate (even if it is the next day). 9. Place the Millicell-CM filters containing the slices onto the 1.1 mL of culture media in the 35-mm petri dishes, and incubate at 36°C in 5% CO2 for 14 to 20 days. Note: The slices thin to optimal thicknesses for immunohistochemistry (IHC) and in situ hybridization histochemistry after incubation for 10 to 20 days. The slices can survive for 1 to 2 months, but may thin too much over such long periods for subsequent experimental manipulations. 10. Media contains penicillin–streptomycin either for the first 3 days in vitro (DIV) or throughout. 11. Media is changed 3 times a week, with fresh media always in a new 35-mm petri dish in order to maximize the vitality and sterility of the culture. 12. In biolistics protocols, we usually shoot the cultured slices after 4 to 5 DIV, replace the medium, and assay by IHC after a total of 10 DIV.
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