1a | First PBS, then 4% PFA | - | - | - | Transcardial perfusion |
1b | 4% PFA | Overnight | 4°C | 15-ml Falcon tube | |
1c | PBS | 30–60 min × 3 times | RT | 15-ml Falcon tube | Mix using a rotary shaker |
Note: Prior to the next step, samples can be transferred to PBS with 0.1% sodium azide and stored long term at 4°C if necessary |
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2a | 100% dH2O | 1 h × 2 | RT | 15-ml Falcon tube | Mix using a rotary shaker |
2b | 20% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
2c | 40% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
2d | 60% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
2e | 80% MeOH in dH2O | 1.5 h | RT | 15 ml Falcon tube | Mix using a rotary shaker |
2f | 100% MeOH | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
2g | 100% MeOH | Overnight | RT | 15-ml Falcon tube | Mix using a rotary shaker |
3a | 66% DCM + 33% MeOH | 2 h | RT | 15-ml Falcon tube | Mix using a rotary shaker and wait till the brain sinks to the bottom |
3b | 66% DCM + 33% MeOH | Overnight | RT | 15-ml Falcon tube | Mix using a rotary shaker and wait till the brain sinks to the bottom |
3c | 100% MeOH | 3 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
4a | 100% MeOH | 3 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
4b | 5% H2O2 in 100% MeOH | Overnight | 4°C | 15-ml Falcon tube | Mix using a rotary shaker |
4c | 80% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
4d | 60% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
4e | 40% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
4f | 20% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
4g | 100% dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
5a | PBS | 1 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
5b | PTx0.5 (PBS with 0.5% Triton X-100) | 1 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
5c | PTx0.5 | 1 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
5d | PTx0.5 | Overnight | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
Note: Prior to the next step, samples can be transferred to PTx0.5 with 0.1% sodium azide and stored for a few days at 37°C if necessary |
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6a | Permeabilization buffer (78.6% PTx0.5 + 1.4% glycine + 20% DMSO) | 48 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker; incubation time can be extended by 24–48 h if needed |
6b | Blocking buffer (84% PTx0.5 + 6% NDS + 10% DMSO) | 48 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker; incubation time can be extended by 24–48 h if needed |
Note: For the next step, we observed that five sequential additions of 1° antibody at 1:1000 dilution (1° Ab booster) helped improve penetration and labeling over a single bolus addition at a 1:200 dilution |
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7a | 1° Antibody (Ab) buffer (92% PTwH0.5 + 3% NDS + 5% DMSO) with 1:1000 1° Ab | 24 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
7b | 1° Ab booster (1:1000 addition of 1° Ab) | 24 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
7c | 1° Ab booster | 24 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
7d | 1° Ab booster | 24 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
7e | 1° Ab booster | 72 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
8a | PTwH0.5 (PBS with 0.5% Tween 20 + 10 μg/ml heparin) | 24 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
8b | PTwH0.5 | 24 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
8c | PTwH0.5 | 24 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
8d | PTwH0.5 | 24 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
Note: Prior to the next step, samples can be transferred to PTwH0.5 with 0.1% sodium azide and stored for a few days at 37°C if necessary; for the next step, we observed that five sequential additions of 2° antibody at 1:500 dilution (2° Ab booster) helped improve penetration and labeling over a single bolus addition at a 1:100 dilution |
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9a | 2° Ab buffer (97% PTwH0.5 + 3% NDS) with 1:500 2° Ab | 24 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
9b | 2° Ab booster (1:500 addition of 2° Ab) | 24 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
9c | 2° Ab booster | 24 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
9d | 2° Ab booster | 24 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
9e | 2° Ab booster | 48 h | 37°C | 1.2-ml Corning cryovial with external threads | Mix using a rotary shaker |
10a | PTwH0.5 | 24 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
10b | PTwH0.5 | 24 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
10c | PTwH0.5 | 24 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
10d | PTwH0.5 | 24 h | 37°C | 15-ml Falcon tube | Mix using a rotary shaker |
Note: Prior to the next step, samples can be transferred to PBS with 0.1% sodium azide and stored long term at 4°C if necessary |
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11a | 100% dH2O | 1 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
11b | 20% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
11c | 40% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
11d | 60% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
11e | 80% MeOH in dH2O | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
11f | 100% MeOH | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
12a | 100% MeOH | 1.5 h | RT | 15-ml Falcon tube | Mix using a rotary shaker |
12b | 66% DCM + 33% MeOH | 2 h | RT | 15-ml Falcon tube | Mix using a rotary shaker and wait till the brain sinks to the bottom |
12c | 66% DCM + 33% MeOH | 2 h | RT | 15-ml Falcon tube | Mix using a rotary shaker and wait till the brain sinks to the bottom |
12d | 66% DCM + 33% MeOH | Overnight | RT | 15-ml Falcon tube | Mix using a rotary shaker and wait till the brain sinks to the bottom |
12e | 100% DCM | 3 h | RT | Wide mouth amber vial with PTFE lined cap | Mix using a rotary shaker and wait till the brain sinks to the bottom |
12f | 100% DCM | 3 h | RT | Wide mouth amber vial with PTFE lined cap | Mix using a rotary shaker and wait till the brain sinks to the bottom |
13a | DBE | 3 h | RT | Wide mouth amber vial with PTFE lined cap | Mix using a rotary shaker and wait till the brain sinks to the bottom |
13b | DBE | 3 h | RT | Wide mouth amber vial with PTFE lined cap | Mix using a rotary shaker and wait till the brain sinks to the bottom |
13c | DBE | 3 h | RT | Wide mouth amber vial with PTFE lined cap | Keep stationary |
14 | Intact brain imaging using LSFM | | | | |