Jul 23, 2020
Brain Clearing (iDISCO)
This protocol is a draft, published without a DOI.
- 1University of California, San Diego
- George Lab @ UCSDTech. support email: olgeorge@ucsd.edu
Protocol Citation: Adam Kimbrough, Olivier George 2020. Brain Clearing (iDISCO). protocols.io https://protocols.io/view/brain-clearing-idisco-822hyge
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 05, 2019
Last Modified: July 23, 2020
Protocol Integer ID: 29498
Guidelines
see idisco.info for more details and information regarding antibodies and protocol
Materials
Solutions:
Note (all of the below solutions contain 0.02% sodium azide—methanol and dcm mixtures do not)
1x PBS
PTx.2 (1L):
50 ml 20x PBS
948 ml ddH2O
2 ml TritonX-100
PtWH (1L):
50 ml 20x PBS
947 ml ddH2O
1 ml heparin (10 mg/ml)
2 ml Tween-20
Permeablization solution (500ml):
400 ml Ptx.2
11.5 g Glycine
100 ml DMSO
Blocking Solution:
42 ml PTx.2
3 ml normal donkey serum
5 ml DMSO
20, 40, 60, 80, 100% methanol
Day 1:
See materials for solutions to mix:
Dehydrate brains with methanol H2O series 1h each wash increasing in concentration (20%, 40%, 60%, 80%, 100%) and once again with 100% (6h total)
Incubate brains in 66% dichloromethane (DCM)/33% methanol overnight
Day 2:
Wash the brains twice for 1h each with 100% methanol to rinse out DCM and then chill samples for 10 minutes at 4 C
Chill 5% hydrogen peroxide in methanol and then incubate the samples overnight at 4 C in the solution
Day 3:
Rehydrate the samples using decreasing concentrations of methanol (1h each): 80%, 60%, 40%, 20%, PBS (5h total)
Wash in Ptx.2 twice for 1h (2h total)
Incubate samples in Permeablization solution overnight at 37 C
Day 4:
Leave samples in Permeabilization solution at 37 C
Day 5:
Incubate samples in Blocking solution at 37 C
Day 6:
Leave samples in Blocking solution at 37 C
Day 7:
Incubate samples in primary antibody (based on what antibody is being used) mixed with 5% DMSO, 3% normal donkey serum in PtwH for 7 days at 37C
Day 14:
Wash samples with PtwH 5x (1h minimum per wash) and leave in the last wash overnight (room temperature)
Day 15: Samples covered with foil from this point forward
Incubate samples in secondary antibody (matched to primary) mixed with 3% normal donkey serum in PtwH for 7 days at 37 C
Day 22:
Wash samples with PtwH 5x (1h minimum per wash) and leave in the last wash overnight (room temperature)
Day 23:
Dehydrate brains with methanol H2O series 1h each wash increasing in concentration (20%, 40%, 60%, 80%, 100%).
Incubate in a fresh solution of 100% methanol and leave samples overnight
Day 24:
Incubate samples for 3h in 66% DCM/33% Methanol
Incubate samples for 15 minutes 2x in 100% DCM to clear off any methanol residue
Move samples to Dibenzyl Ether without shaking for long term storage
Store at room temperature in the dark until imaged