1. Regent prepares
a) 1 M MgCl2
10.165g MgCl2·6H2O dissolved in 50 mL H2O.
b) 1 M CaCl2
7.35g CaCl2·2H2O into 50 mL H2O.
c) 0.1 M NaHCO3
1.68g NaHCO3 dissolved into 200 mL H2O.
d) Solution A
| In 200 mL H2O | |||
| 0.23 M | Sucrose | Sucrose | 21.92 g |
| 1 mM | MgCl2 | 1 M MgCl2 | 0.2 mL |
| 0.5 mM | CaCl2 | 1 M CaCl2 | 0.1 mL |
| 1 mM | NaHCO3 | 0.1 M NaHCO3 | 2 mL |
e) Solution B
| In 100 mL H2O | |||
| 0.23 M | Sucrose | Sucrose | 10.96 g |
| 1 mM | NaHCO3 | 0.1 M NaHCO3 | 1 mL |
f) 1.0 M sucrose
1 mL 0.1 M NaHCO3, 34.25g sucrose in H2O make up to 100 mL.
g) 1.4 M sucrose
1 mL 0.1 M NaHCO3, 47.9g sucrose in H2O make up to 100 mL.
2. step by step
a) Perfusing mouse with cold PBS, decapitation and quick dissection hippocampus on ice. Frozen and keeping samples in -80 until extraction.
b) Chop samples into 8 mm2 small pieces with scissors.
c) Add 4-fold vol (mL/g) solution A. Keep on ice at least 20 min.
d) Homogenize at 1000 rpm with 6 or 7 up-and-down motions.
e) Transfer samples to new tubes and dilute to 10-flod vol (mL/g) with solution A.
f) 1400g * 10 min. Save supernatant.
g) Dilute pellet with solution A to 4-fold vol (mL/g). Do step c) to e). 710g * 10 min. Save supernatant. Pellet is P1.
h) Combine supernatants in step f) and g). 710g * 10 min.
i) Collect supernatant (S1), 13800g * 10 min. Supernatant and pellet obtained in this step are S2 and P2, respectively.
j) Resuspend the P2 with solution B gently (~2.5-fold vol). Layer the suspension on gradients composed of 1.0 and 1.4 M sucrose, and centrifuge at 82500g for 70 min.
k) Collect the bands in the interface between 1.0 and 1.4 M sucrose layer, measure volume and concentration.