TITLE: Human Brain Sequential Extraction (Tau)
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol details Human Brain Sequential Extraction (Tau). This protocol is an adaptation of the work of several labs.

[STEPS]
SECTION: 1 Day Before Extraction
1.   

SECTION: 1 Day Before Extraction
2. Make sure glass 100 or 40 mL homogenizers and pestles (A and B) are cleaned with 70%
ethanol, wrapped in foil, and autoclaved.
SECTION: 1 Day Before Extraction
3.  Clean out ultracentrifuge tubes and caps with 70% ethanol and dry.
SECTION: 1 Day Before Extraction
4. Transfer brain tissue to -20 °C freezer the night before (speeds up thaw).

SECTION: 1 Day Before Extraction
5.  Ensure there are sufficient protease inhibitor, phosphatase inhibitor, and DTT.
SECTION: Day 1 - Preparation
6. Turn on the ultracentrifuge (Optima L-100 XP), set the temperature for 4 °C and turn the vacuum on. Make sure Ti-45 and Ti-70 rotors are available. They may be in room 5124.

SECTION: Day 1 - Preparation
7.  Fill 2 buckets with wet ice.
SECTION: Day 1 - Preparation
8. Warm PMSF to Room temperature.

SECTION: Day 1 - Preparation
9. Record information about the tissue to be extracted. Note the weight of the dish to be used for
weighing the gray matter.
SECTION: Day 1 - Preparation
10.  Fill a 15 mL conical with 10% neutral buffered formalin (NBF) and label with case number.
SECTION: Day 1 - Extraction
11. Thaw bag(s) of brain tissue on ice.

SECTION: Day 1 - Extraction
12.  Move brain to petri dish and weigh it.
SECTION: Day 1 - Extraction
13. Once tissue is thawed, remove meninges and blood vessels. Cut one thin, representative piece
off and transfer it to 10% neutral buffered formalin for fixation and subsequent IHC.
SECTION: Day 1 - Extraction
14. Carefully resect the remaining gray matter from white matter in ice-cold PBS. Transfer gray matter
to a petri dish on ice to be weighed.

SECTION: Day 1 - Extraction
15. Weigh gray matter.
SECTION: Day 1 - Extraction
16. Prepare 30 mL Extraction Buffer per gram gray matter. Add the following to ice-cold Base
Buffer: cOmplete protease inhibitor (1 tab/50 mL), phosphatase inhibitors (2&3) (1:100), 0.1 millimolar (mM) PMSF (1:5000), 2 millimolar (mM) DDT (1:500). 

SECTION: Day 1 - Extraction
17. Add 9 volumes of Extraction Buffer to the homogenizer tube. Cut gray matter into small bits,
and use a buffer to transfer these bits to the tube. 
SECTION: Day 1 - Extraction
18. Homogenize with pestle A until the pestle moves easily. 
SECTION: Day 1 - Extraction
19. Homogenize with pestle B 3x 25 strokes. Save 200 µL of this as Total Homogenate. 

SECTION: Day 1 - Extraction
20. Pour homogenate evenly into Ti-45 tubes (fits ~50 mL/tube). Balance tubes to within 0.1 g. 

SECTION: Day 1 - Extraction
21. Spin at 11300 rpm in the Ti-45 (10000 x g) for 10 min at 4 °C. 

SECTION: Day 1 - Extraction
22. Pour supernatant into 50 mL conical by filtering it through a piece of kimwipe folded into two
layers placed in a funnel. 

 

SECTION: Day 1 - Extraction
23. Add 9 volumes Extraction Buffer to the centrifuge tube and homogenizer. Transfer pellet in
buffer to homogenizer. 
SECTION: Day 1 - Extraction
24. Homogenize with pestle A until the pestle moves easily. 
SECTION: Day 1 - Extraction
25. Homogenize with pestle B 3x 25 strokes.
 
 

SECTION: Day 1 - Extraction
26. Pour homogenate evenly into Ti-45 tubes (fits ~50 mL/tube). Balance tubes to within 0.1 g.

SECTION: Day 1 - Extraction
27. Spin at 11300 rpm in the Ti-45 (10000 x g) for 10 min at 4 °C. 

SECTION: Day 1 - Extraction
28. During the spin, transfer Sup 1 to a beaker with a stir bar. Add sarkosyl to a final 1%. 
SECTION: Day 1 - Extraction
29. Pour supernatant into 50 mL conical by filtering it through a piece of kimwipe folded into two
layers placed in a funnel. 

 

SECTION: Day 1 - Extraction
30. Combine Sup 1 and Sup 2 in a beaker with a stir bar. Add sarkosyl to a final 1% concentration
(1/27). 

 

SECTION: Day 1 - Extraction
31. Stir Total Sup for 1- 90 min at Room temperature.

SECTION: Day 1 - Extraction
32. Add the Extraction Buffer to the centrifuge tube and transfer the pellet to the homogenizer. You can use the same vol as Sup 2 for homogenization. 

 

SECTION: Day 1 - Extraction
33. Add Total Sup to Ti-45 tubes. Balance tubes to within 0.1 g. Spin at 40000 rpm in the Ti-45 (125000 x g) for 75 min at 4 °C. 

SECTION: Day 1 - Extraction
34. Pipet out supernatant into a beaker. Myelin will have floated. Remove this with a pipette. 

 

SECTION: Day 1 - Extraction
35. Use the vacuum to aspirate remaining liquid around the Sark Pellet.

 

SECTION: Day 1 - Extraction
36. Add 10 mL DPBS to each centrifuge tube to wash the Sark Pellet. Use the vacuum to remove DPBS. 

SECTION: Day 1 - Extraction
37. Add 2 mL DPBS to wash Sark Pellet a second time. Use vacuum to remove DPBS.

SECTION: Day 1 - Extraction
38. Add 1 mL DPBS to each tube. Pipette liquid towards the Sark Pellet with a cut P1000 tip until the
pellet has loosened. Use a transfer pipette to transfer the pellet to a Ti-70 centrifuge tube. 

 

SECTION: Day 1 - Extraction
39. Use a cut P1000 tip to pipette up and down to resuspend the pellet. 
SECTION: Day 1 - Extraction
40. Add DPBS into the centrifuge tube to reach maximum volume. Balance tubes to within 0.1 g. 

SECTION: Day 1 - Extraction
41. Spin Sark Pel at 50000 rpm in the Ti-70 (18000 x g) for 30 min at 4 °C.

SECTION: Day 1 - Extraction
42. Save 200 µL of the supernatant as DPBS wash. Remove the remaining supernatant by vacuum. 

SECTION: Day 1 - Extraction
43. Add 100 µL DPBS/1 g tissue to the pellet. Use a cut P1000 tip to first loosen the pellet, then
break it up as much as possible without pipetting up and down. Transfer to 1.5 mL tubes. 


 

SECTION: Day 1 - Extraction
44. Vortex briefly and spin in a tabletop centrifuge at 1000 x g for 1 min. 

SECTION: Day 1 - Extraction
45. Rock the tube at 4 °C  . 

SECTION: Day 1 - Cleanup
46. Bleach all homogenizers, tools and tubes, but use only soap for metal lids. 
SECTION: Day 1 - Cleanup
47. Rinse centrifuge tubes for 10 min after bleaching. 

SECTION: Day 1 - Cleanup
48. Bleach out ice bucks. 
SECTION: Day 1 - Cleanup
49. Bleach vacuum flask and rinse out. 
SECTION: Day 2
50. Move fixed brain to leaching buffer and cassette for paraffin embedding. 
SECTION: Day 2
51. Spin in a tabletop centrifuge at 1000 x g for 1 min at 4 °C.

SECTION: Day 2
52. Pass the suspension repeatedly through a 27G ½ needle to homogenize. 

 

SECTION: Day 2
53. Transfer the resuspended Sark Pel to an autoclaved 1.5 mL Beckman ultracentrifuge tube. 
SECTION: Day 2
54. Add 100 µL DPBS to the centrifuge tube and resuspend well. 

 

SECTION: Day 2
55. Balance tubes to within 0.1g. Spin in OptimaMAX-TL (room 5124) (TLA100.3 rotor with adaptors) at 45000 rpm (80000 x g) for 30 min at 4 °C. 

SECTION: Day 2
56. Remove supernatant and add 100 µL DPBS/1 g tissue to the pellet. 

SECTION: Day 2
57. Vortex to mix. Freeze or continue to process Sark Pellet. 
SECTION: Day 2 or 3 – Further Purification
58. Thaw the Sark Pellet. 
SECTION: Day 2 or 3 – Further Purification
59. Sonicate the Sark Pellet with 20x 1 sec pulses with hand sonicator. 
SECTION: Day 2 or 3 – Further Purification
60. Balance tubes to within 0.1g. Spin in OptimaMAX-TL (room 5124) (TLA100.3 rotor with
adaptors) at 45000 rpm (80000 x g) for 30 min at 4 °C. 

SECTION: Day 2 or 3 – Further Purification
61. Transfer supernatant into another tube as High g Sup using sterile pipette tips.
SECTION: Day 2 or 3 – Further Purification
62. Resuspend the pellet with 20% of the volume of DPBS (e.g. for 700 µL Sup, add 140 µL). 

SECTION: Day 2 or 3 – Further Purification
63. Sonicate High g Pellet for 90-120x 1 s pulses with hand sonicator on ice to break up the pellet.

 


SECTION: Day 2 or 3 – Further Purification
64. Transfer the resuspended High g Pellet to a 1.5 mL tube. Spin in tabletop centrifuge at 10000 x g for 30 min at 4 °C. 

SECTION: Day 2 or 3 – Further Purification
65. Transfer supernatant using a sterile pipette tip to a 1.5 mL tube. 

 


SECTION: Day 2 or 3 – Further Purification
66. Add an equal volume of DPBS to the pellet and sonicate 30x 1 s pulses with hand sonicator to resuspend the pellet.