Isolation of tissue
- Hippocampi are rapidly dissected from the brain in 2 mL HIBERNATE A supplemented with B27 (Cat. No. 17504) and 0.5 mM L-glutamine (Cat. No. 25030) at 4°C in a 35 mm diameter dish.
- Meninges and excess white matter are removed in the same medium to a second dish at 4°C.
- Hippocampi are transferred to sterile paper prewet with the same medium on the cooled stage of a MacIlwain tissue chopper.
- 0.5 mm thickness slices are made perpendicular to the long axes of the hippocampi and transferred to a tube at 4°C of the same medium.
- After shaking for 8 minutes at 30°C, slices are transferred with a wide bore pipette to another tube at 30°C containing papain.
Digestion of tissue
- Papain (Worthington 3119, 15-23 U/mg protein and not activated by cysteine) is prepared by dissolving 12 mg in 6 mL of HIBERNATE-A, warming for 5 minutes at 37°C and then filter sterilizing. The papain solution should be used within 3 hours.
- Slices are incubated for 30 minutes in a 30°C water bath with a platform rotating at a speed sufficient to suspend the slices (e.g.. 170 rpm).
- Slices are transferred to a 15 mL tube containing 2 mL of HIBERNATE-A/B27 at 30°C and allowed to sit for 5 minutes at room temperature.
- Slices are triturated 10 times (in about 30 seconds) with a siliconized 9 inch Pasteur pipette with the tip fire polished to an opening of 0.7 to 0.9 mm diameter.
- The pieces are allowed to settle for 2 minutes and the supernatant transferred to another tube.
- The sediment from the first tube is resuspended in 2 mL HIBERNATE-A/B27