TESOS 3 protocol for whole brain (edited by Jiayu Wang)
Materials:
Quadrol
CAS: 102-60-3
Sigma-Aldrich, 122262
Tert-Butanol
CAS: 75-65-0
Sigma-Aldrich, 471712
TritonX100
CAS: 9036-19-5
Sigma-Aldrich, 2315025
Urea
CAS: 57-13-6
Aladdin, U111902
DMBA
3,4-Dimethoxybenzyl Alcohol
CAS: 93-03-8
Sigma-Aldrich, 188794
GMA
2-Hydroxyethyl methacrylate
CAS: 868-77-9
Sigma-Aldrich, 128635
DA200
Polyethylene glycol 200
CAS: 25322-68-3
Aladdin, P109707
Irgacure2959
2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone
CAS: 106797-53-9
Sigma-Aldrich, 410896
Bis-Acrylamide
N,N’-Methylenebisacrylamide
CAS: 110-26-9
Aladdin, M128783
Acrylamide
Macklin, A800656
Solutions:
25% w/v Quadrol solution
TESOS 3 delipidation medium——TTU：
60% v/v Tert-Butanol +10% w/v TritonX100 +15% w/v urea +1% w/v Quadrol
TESOS 3 clearing medium:
DMBA(v/v) |
40% |
40 |
mL |
GMA(v/v) |
30% |
30 |
mL |
DA200(v/v) |
15% |
15 |
mL |
H2O(v/v) |
15% |
Up to100 |
mL |
Irgacure2959(w/v) |
1% |
1 |
g |
Urea(w/v) |
15% |
15 |
g |
Acrylamide(w/v) |
10% |
10 |
g |
Bis(w/v) |
4% |
4 |
g |
Total volume |
100 |
mL |
Attention: add the solid part after adding liquid to 100 mL.
Procedures:
1. Perfusion to sacrifice mice and harvest samples: heart perfuse with PBS and then 4% PFA to wash away as much blood as possible.
The indication of a successful heart perfusion is the liver turning pale.
2. Fixation: fix samples with 4%PFA at 4°C overnight.
3. Decolorization:
--Wash the sample with distill water to remove the remaining PFA.
--Place whole organs into 25% Quadrol for 2 days at 37°C under constant shaking (~70rpm).
--Refresh medium daily.
4. Delipidation:
--Place whole organs into TTU medium for 6 days at 37°C under constant shaking (~70rpm).
--Refresh the medium once every two days.
-- Place whole organs into TESOS 3 clearing medium under constant shaking (~70 rpm) until transparency is reached.
6. polymerization: UV light (after polymerization, put the gel into 30°C oven for two days for better polymerization)
7. Imaging
