TESOS2 protocol (edited by Jiayu Wang)

Materials:

Quadrol

CAS: 102-60-3

Sigma-Aldrich,122262

Tert-Butanol

CAS: 75-65-0

Sigma-Aldrich, 471712

BB

CAS: 120-51-4

Sigma Aldrich, W213802

BED

CAS: 64401-02-1

Sigma Aldrich, 413550

EGDMA

CAS: 97-90-5

Sigma Aldrich, 335681

V65 (thermal initiator)

CAS: 4419-11-8

Aladdin, A189143

Irgacure2959 (UV initiator)

2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone

CAS: 106797-53-9

Sigma-Aldrich, 410896

Solutions:

25% w/v Quadrol solution (Quadrol is very vicious and weighing is easier)

30% v/v tert-Butanol + 5% w/v Quadrol

50% v/v tert-Butanol + 5% w/v Quadrol

70% v/v tert-Butanol + 5% w/v Quadrol

Dehydration solution:

TBQ: 75% v/v Tert-butanol + 25% v/v pure Quadrol

TESOS2 clearing medium:

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 (~70 rpm).

--Refresh medium daily.

4. Delipidation:

--Place whole organs into 30TB-50TB-70TB medium (each medium for 1 day) at 37°C under constant shaking (~70 rpm).

5. Dehydration:

--Place whole organs into TBQ solution for 2 days at 37°C under constant shaking (~70 rpm).

--Refresh medium daily.

6. Clearing and polymerization:

Method A:

--Add 1% UV initiator (Irgacure2959) into the transparent solution with sample and shake it at 37°C for 1 day (~70 rpm).

--Put the sample into the silicone embedding box, and add clearing medium containing 1% UV initiator until cover the sample.

--polymerize with UV light.

Method B:

--Add 0.5% thermal initiator (v65) into the transparent solution with the sample and shake it at 4°C for four days (~70 rpm).

--Put the sample into the silicone embedding box, and add clearing medium containing 0.5% v65 initiator until cover the sample.

--Degassing by vacuum for 2-3 hours.

--Put the silicone embedding box with the sample and clearing medium into a 37°C water bath, and heat the silicone embedding box for several hours until the clearing medium finish polymerization.

--Put the gel under UV light for better polymerization.

7. Imaging.

Figures of TESOS2 in detail

Silicone embedding box

Vacuum pumping device

37°C water bath device (the silicone embedding box with clearing medium was

covered by a coverslip on the top to make sure the smooth surface of the gel).

polymerize with UV light (when the clearing medium is under polymerization,

cooling the silicone embedding box on ice).