Start by fire-polishing three pasture pipettes using a bunsen burner, creating decreasing diameters. Next, sterilize the abdomen of a euthanized pregnant mouse dam with 70%ethanol. Cut open the abdominal cavity from the pubic synthesis to the xiphoid process of the rib cage.
Now, extract the uterine horn from the abdominal cavity and place it in a 100-millimeter plate filled with ice-cold HBSS solution. Extract and separate all embryos from the washed uterus using fine forceps. Quickly decapitate the extracted mice embryos and place the heads in a 60-millimeter Petri dish filled with HBSS.
Place a pair of fine forceps in the eye cavity to hold the brain. With another pair, peel the skin and skull until the brain is visible. Scoop the brain out of the olfactory bulbs from the skull and flip it upside down.
Observe the cortex on the ventral side and the hypothalamus on the dorsal side. Use curved forceps to remove the layer of meninges and blood vessels until the brain appears white and clear, and separate the hypothalamic area from the rest of the brain. Cut the hypothalamus into three to four small fine pieces, and use a pipette to transfer the pieces into a 15-milliliter tube.
Next, fill the tube with six milliliters of HBSS. Then add Enzyme Mix 1 once the tissue has settled and the supernatant removed. Incubate the tube in a 37-degree Celsius water bath for 15 minutes, agitating every five minutes to re-suspend the tissue.
Now, add 30 microliters of Enzyme Mix 2 and dissociate the tissue by pipetting up and down 10 times with a large diameter pasture pipette. After an additional 10-minute incubation, add the remaining 15 microliters of Enzyme Mix 2. Utilizing two fire polished pipettes of decreasing diameter, pipette the tissue 10 times.
Immediately add 10 milliliters of HBSS with 0.5%BSA to the tube. Then centrifuge the tube at 300 G for 10 minutes at room temperature. Aspirate the supernatant and re-suspend the cell pellet in a one milliliter of HBSS with 0.5%BSA.
