Protocol of the PEGASOS Method

PEGASOS passive immersion protocol for clearing adult mice organs

Materials:

Quadrol; Sigma-Aldrich; 122262

Tert-Butanol; Sigma-Aldrich; 471712

PEG-MMA500; Sigma-Aldrich; 447943

Benzyl Benzoate; Sigma-Aldrich; B6630

EDTA (Ethylenediaminetetraacetic acid disodium salt dehydrate); Sigma-Aldrich; E5134

Glass vials or 50ml conical tubes

Solutions:

0.5M EDTA (pH=7.0-7.5)

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

3% ammonium solution

30% v/v tert-Butanol + 3% w/v Quadrol

50% v/v tert-Butanol + 3% w/v Quadrol

70% v/v tert-Butanol + 3% w/v Quadrol

[70% v/v tert-Butanol + 30% v/v PEG-MMA-500] + 3% w/v Quadrol

BB-PEG clearing solution (R.I. 1.543): [75% v/v Benzyl Benzoate + 25% v/v PEG-MMA-500] + 3% w/v Quadrol

Procedures:

Samples can be placed in 50ml conical tubes for treatment in each step.

1. Perfusion to sacrifice mice and harvest samples: transcardiac perfuse with PBS and then 4% PFA to wash away as much blood as possible.

The indication of a successful transcardiac perfusion is the liver turning pale.

2. Fixation: fix samples with 4%PFA at 4°C overnight.

3. Decalcification: (for hard tissue only)

Immerse samples in 0.5M EDTA and place in a 37°C shaker (~100rpm) for 2-4 days with daily medium change. The duration of decalcification depends on the size of the bone. Usually, an adult mouse femur or mandible requires 4 days for complete decalcification.

4. Decolorization:

--Place whole organs into 25% Quadrol for 1-2 days at 37°C under constant shaking (~100 rpm).

--Refresh medium daily.

--(optional) 3% ammonium solution 6 hours for heavily colorized samples that were not perfused.

5. (Optional) Whole mount immunofluorescent staining of samples.

6. Delipidation and dehydration will be performed in 37°C shaker (~100rpm). See the table below for the recommended duration for each step.

a. 30% tert-Butanol + 3% Quardol

b. 50% tert-Butanol + 3% Quardol

c. 70% tert-Butanol + 3% Quardol

d. [70% tert-Butanol + 30% PEG-MMA-500]+ 3% Quardol

7. Clearing:

Briefly rinse the sample with the BB-PEG clearing medium and then immerse and shake it at 37°C (~100rpm) until transparency is reached.

Tip: Freshly made BB-PEG solution is colorless and turns slightly yellowish after exposing in air. Somehow, we noticed that BB-PEG exposed in air for 12 hours achieved better transparency than fresh BB-PEG.

The optimal duration for processing samples of different tissue types and sizes should be determined by experiments. The following table provides recommended times for different samples.

8. Storage: Samples can then be preserved in the clearing medium covered with aluminum foil at room temperature for a long term.