Table 3. iDisco+tissue clearing steps.
For primary and secondary antibody concentration, please refer to Tables 1 and 2. RT, room temperature; DCM, dichloromethane; DBE, dibenzyl ether; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; PTx2, 0.05% Triton X-100 in 1× PBS; PTwh, 0.0.5% Tween 20, 0.5 ml of heparin (10 mg/ml) in 1× PBS.
| Process | Condition | Reagent | Solvent |
| Initial wash | 15 min 3× in each | PTx2 and PTwh | 1× PBS |
| Serial dehydration | 1 hour each | 20/40/60/80/100% methanol | 1× PBS |
| Defatting | Overnight | DCM (66%, v/v) | Methanol |
| Bleaching | Overnight | H2O2 (5%, v/v) | Methanol |
| Serial rehydration | 1 hour each | 20/40/60/80/100% PBS | Methanol |
| Permeabilization | 4 days | Triton X-100, 20% DMSO | 1× PBS |
| Blocking | 2 days | Goat serum (6%, v/v) and DMSO (10%, v/v) |
PTx2 |
| Primary antibody incubation | 7 days, 37°C, 100 rpm | Goat serum (3%, v/v) and DMSO (5%, v/v) |
PTx2 |
| Wash | Overnight | PTwh | |
| Secondary antibody incubation | 6 days, 37°C, 100 rpm | Goat serum (3%, v/v) and DMSO (5%, v/v) |
PTx2 |
| Final dehydration | 1 hour each | 20/40/60/80/100% methanol | DiH2O |
| Defatting | 3 hour, RT | DCM (66%, v/v) | Methanol |
| Defatting | Overnight | DCM 100% | DCM |
| Final defatting/clearing | 30 min—hold indefinitely | 100% DBE | DBE |