Materials and Reagents

1. 1.5 ml Eppendorf tube (Eppendorf, for 1 ml solution)
2. 5 ml Eppendorf tube (Eppendorf, catalog number: 0030119401 ; optional for 3 ml solution, for thick brain slices or whole-mount samples)
3. Paint brush (for thin brain slices, No. 1-2, see Figure 1)
4. Silicone rubber sheet (translucent, 0.2 mm thick; e.g., AS ONE, catalog number: 6-9085-13 ; see Figure 1)
   Note: Various thickness of silicone rubber sheets are available from Togawa Rubber (AS ONE), Professional Plastics, CS Hyde, etc., ranging from 0.1 mm to 8.0 mm. The thickness should match that of brain slices.
5. Glass slide (76 mm x 26 mm; MATSUNAMI Glass; Figure 1)
6. Coverslips (18 x 18 mm, No 1.5H; e.g., Paul Marienfeld, catalog number: 0107032 or ZEISS, catalog number: 474030-9000-000 ; Figure 1)
   Note: No 1.5H (170 ± 5 μm thick) is highly recommended for super-resolution imaging. 
7. Sodium chloride (NaCl)
8. Sodium hydrogen phosphate dodecahydrate (Na₂HPO₄•12H₂O)
9. Potassium chloride (KCl)
   
10. Potassium dihydrogen phosphate (KH₂PO₄)
11. 4% paraformaldehyde (PFA; e.g., NACALAI TESQUE, catalog number: 26126-25 ) in PBS
12. 20% Saponin (NACALAI TESQUE, catalog number: 30502-42 ) in ddH₂O with filter sterilization
    Note: Different vendors prepare saponin from different species of plants. We strongly recommend NACALAI TESQUE. Brownish lots (often found in Sigma-Aldrich) should be avoided.
13. Low-melting point agarose (e.g., Thermo Fisher Scientific, catalog number: 16520100 )
14. Omnipaque 350 (e.g., DAIICHI SANKYO, Omnipaque 350 Injection, 50 ml in 1 bottle; also available from GE healthcare)
15. Histodenz (e.g., Sigma-Aldrich, catalog number: D2158 )
16. Sodium azide (e.g., Sigma-Aldrich, catalog number: 13412 )
17. 1 M stock of Tris-HCl, pH 7.6 (e.g., NACALAI TESQUE, catalog number: 35436-01 ), used to prepare Tris-EDTA buffer
18. 0.5 M stock of EDTA, pH 8.0 (e.g., Dojindo, catalog number: 347-07481 ), used to prepare Tris-EDTA buffer
19. Immersion media:
    Glycerol (e.g., Type G Immersion Liquid, Leica Microsystems, catalog number: 11513910 ; Glycerine solution, Leica Microsystems, catalog number: 11513872 ) for SeeDB2G
    Oil (Type F, Olympus, MOIL-30; also available from Leica, Zeiss, etc.) for SeeDB2S
20. (Optional) SeeDB2 Trial Kit (Wako Pure Chemical Industries, catalog number: 294-80701 ) 
21. Phosphate-buffered saline (PBS) (see Recipes)
22. Tris-EDTA buffer, pH 7.6 (see Recipes)
23. Permeabilization solution (see Recipes)
24. Solution 1 (see Recipes)
25. Solution 2 (see Recipes)
26. SeeDB2 solutions (see Recipes)
    
    1. SeeDB2G with saponin (clearing)
    2. SeeDB2G (mounting)
    3. SeeDB2S with saponin (clearing)
    4. SeeDB2S (mounting)
    
    
    Figure 1. Materials required for preparing imaging chamber and slice mounting

Equipment

1. Perforated spoon (optional for handling thick brain slices, custom-ordered, flat head, head diameter = 15 mm, see Figure 1 and Video 1)
2. Ring forceps (optional for whole-mount samples, e.g., Natsume Seisakusho, NAPOX, catalog number: A-26 , see Figure 1)
3. Vibratome (e.g., LinearSlicer, DOSAKA, model: PRO7N )
4. Seesaw shaker (e.g., Bio Craft, model: BC-700 )
5. Rotator (e.g., TAITEC, model: RT-30mini, catalog number: 0057154-000 )
6. Fluorescence microscope
   
   1. Confocal microscope (e.g., Olympus, model: FV3000 ; Leica Microsystems, model: Leica TCS SP8 ; Nikon, model: A1+ )
   2. Super-resolution microscope (e.g., Zeiss, model: LSM 880 with Airyscan; Leica Microsystems, model: Leica TCS SP8 with HyVolution 2; Leica Microsystems, model: Leica TCS SP8 STED )
7. Objective lenses
   Examples: 63x oil-immersion (NA = 1.4, WD = 0.14 mm) (Leica Microsystems, model: HC PL APO 63x/1.4 Oil CS2, catalog number: 15506350 ); 100x oil-immersion (NA = 1.4, WD = 0.13 mm) (Leica Microsystems, model: HC PL APO 100x/1.4 Oil CS2, catalog number: 15506325 ); 63x glycerol-immersion (NA = 1.3, WD = 0.28 mm) (Leica, model: HCX PL APO 63x/1.30 GLYC CORR, catalog number: 11506193 ); 63x oil-immersion (NA = 1.4, WD = 0.19 mm) (Carl Zeiss, model: Plan-Apochroamt 63x/1.40 Oil DIC, catalog number: 440762-9904-000 )
   

Procedure