Materials and Reagents

1. Phosphate-buffered saline (PBS)
   
2. 4% paraformaldehyde (PFA) in PBS
   
3. D(-)-Fructose (≥99%) (e.g., Sigma-Aldrich, catalog number: F0127-500G )
   
4. α-thioglycerol (≥95%) (e.g., Sigma-Aldrich, catalog number: M1753-100ML )
   
5. 2,2’-thiodiethanol  (≥99%) (e.g., Sigma-Aldrich, catalog number: 166782-500G ) (optional)
   
6. Glycerol (≥99%) (e.g., Sigma-Aldrich, catalog number: G9012-500ML ) (optional)
   
7. Low melting point agarose (e.g., Life Technologies, catalog number: 16520 -100) (optional)
   
8. SeeDB (see Recipes)
   
9. SeeDBp (see Recipes)
   
10. Immersion solution (for commercial objective lenses) (see Recipes)
    
11. Immersion solution (for customized objective lens for SeeDB) (see Recipes)
    

Equipment

1. 50 ml conical centrifuge tube (e.g., BD Biosciences, Falcon^®) (for whole- and hemi-brain samples)
   
2. Culture dish (30 or 60 mm diameter) (e.g., BD Biosciences, Falcon^®) (for slice preparation and fragile samples)
   
3. Overhead tube rotator (recommended for clearing large samples) (Figure 1A)
   
4. Seesaw shaker (for fragile samples) (Figure 1B)
   
   
   Figure 1. Equipment used for optical clearing. For efficient optical clearing, it is important to shake samples in fructose solutions. We recommend using 50 ml conical centrifuge tube and overhead rotator for clearing large samples (e.g., whole- or hemi-brain samples). Fragile samples (e.g., brain slices) can be cleared in culture dishes and seesaw shaker. A. Overhead tube rotator, B. Seesaw shaker
   
   
5. Air incubator (optional, for SeeDB37 and SeeDB37ht protocols)
   
6. Coverslips (24 mm x 60 mm for small preparation and slices; 50 mm x 70 mm for whole-mount samples) (e.g., Matsunami Glass, catalog number: C050701 )
   
7. Handmade glass bottom Petri dish (100 mm diameter) or a commercial glass bottom dish (MatTek, catalog number: P100G-1.5-30-F )
   
   
   Figure 2. Imaging chambers for fluorescence microscopy. A. An imaging chamber for inverted microscopes. B. An imaging chamber for upright microscopes with short working distance objective lenses. C. An imaging chamber for upright microscopes with long working distance objective lenses. We use glass bottom Petri dish to keep immersion solutions.
   
   
8. Silicone rubber sheet (optimal thickness should be chosen. For example, 6-mm-thick silicone sheet is recommended for 6-mm thick adult mouse brain.) (e.g., TOGAWA RUBBER, catalog number: K-125 )
   Note: Silicone rubber sheets adhere to the coverslips and Petri dishes without any adhesives.
   
9. Thermo plate (optional, only for samples cleared with SeeDB37 or SeeDB37ht) (e.g., Tokai Hit)
10. Fluorescence microscope
    Confocal microscope (for imaging up to 1-2 mm depth)
    Multiphoton microscope (recommended for deeper imaging)
    
11. Objective lenses (examples)
    10x air (NA=0.4, WD=3.1 mm) (OLYMPUS, model: UPLSAPO10X2 )
    10x water immersion (NA=0.3, WD=3.5 mm) (OLYMPUS, model: UMPLFLN10xW )
    25x water immersion (NA=1.05, WD=2 mm) (OLYMPUS, model: XLPLN25XWMP )
    25x scale immersion (NA=1.0, WD=4 mm) (OLYMPUS, model: XLPLN25SVMP )
    Water-immersion lenses perform much better than air-immersion objective lenses because spherical aberrations are smaller (Figure 5). In the two-photon microscopy, we also use a custom-made objective lens that performs best under the refractive index of SeeDB (~1.49). Please contact Olympus for its availability.
    

Procedure