Materials and Reagents

1. Chloral hydrate
   
2. Instant glue
   
3. Biotinylated dextran amine (BDA) (3,000 molecular weight, 10% solution in PBS) (Life Technologies, Invitrogen^TM, catalog number: D-7135 )
   
4. Alexa Fluor 488-conjugated cholera toxin subunit b (Alexa488-CTb, 0.5% solution in saline) (Life Technologies, Invitrogen^TM, catalog number: C-34775 )
   
5. Distilled H₂O (dH₂O)
   
6. Pentobarbital
   
7. Sodium azide
   
8. Phosphate-buffered saline (PBS)
   
9. Alexa Flour 594-conjugated streptavidin (Life Technologies, Invitrogen^TM, catalog number: S-11227 )
   
10. Normal donkey serum (10% solution in PBS) (Jackson Immuno Research, catalog number: 017-000-121 )
    
11. Primary antibodies for immunofluorescence
    Note: We almost use self-made antibodies (Kudo et al., 2012).
    
12. Secondary antibodies
    Note: We use products from Invitrogen and/or Jackson Immuno Research, whose host are donkey.
    
13. Acetic anhydrate
    
14. Triethanolamine-HCl
    
15. Formadime
    
16. Tris(hydroxymethyl) aminomethane (TRIS)
    
17. Ficoll
    
18. Polyvinylpyrrolidone
    
19. Bovine serum albumin (BSA)
    
20. tRNA
    
21. Ethylenediaminetetraacetic acid (EDTA)
    
22. N-Lauroylsarcosine sodium salt (NLS)
    
23. Dextran sulfate
    
24. cRNA probes (Kudo et al., 2012; Yamasaki et al., 2010)
    
25. Sodium citrate
    
26. Lodoamide
    
27. DIG blocking reagent (Roche Applied Science, catalog number: 11096176001 )
    
28. Normal sheep serum (Chemicon, catalog number: S22-100ML )
    
29. Maleic acid
    
30. Tyramide signal amplification (TSA) blocking reagent (PerkinElmer Life and Analytical Science, catalog number: FP1020 )
    
31. Peroxidase-conjugated anti-DIG antibody (1:1,000 in DIG blocking buffer) (Roche Diagnostics, catalog number: 11207733910 )
    
32. Cyanine 3 (Cy3) Amplification Reagent (PerkinElmer Life and Analytical Science, catalog number: FP1170 )
    
33. 1x Plus Amplification Diluent (PerkinElmer Life and Analytical Science, catalog number: FP1135 )
    
34. H₂O₂
    
35. Anti-Alexa Fluor 488 antibody for detection of Alexa488-CTb (raised in rabbit) (1:1,000 dilution) (Invitrogen, catalog number: A-11094 )
    
36. TOTO-3 iodide (642/660) (1:50 in PBS) (Life Technologies, Invitrogen^TM, catalog number: T3604 )
    
37. Primary antibody solution (1 μg/ml in PBS-T)
    
38. Fluorophore-linked secondary antibody solution (1:200 in PBS-T)
    
39. Digoxigenin (DIG)-labeled cRNA probes
    
40. Dimethyl sulfoxide (DMSO)
    
41. Blocking reagent (Roche Applied Science, catalog number: 11096176001)
    
42. Saline (see Recipes)
    
43. 3.5% chloral hydrate (see Recipes)
    
44. 0.1 M PB (see Recipes)
    
45. 5 N NaOH (see Recipes)
    
46. 4% Paraformaldehyde (PFA) (see Recipes)
    
47. 20% Tween-20 (see Recipes)
    
48. PBS containing 0.1% Tween-20 (PBS-T) (see Recipes)
    
49. 0.25% acetic anhydrate in 0.1 M triethanolamine-HCl (pH 8.0) (see Recipes)
    
50. 1 M Tris-HCl (pH 7.4 or 8.0) (see Recipes)
    
51. 5 M NaCl (see Recipes)
    
52. 0.5 M ethylenediaminetetraacetic acid (EDTA) (see Recipes)
    
53. Hybridization buffer (see Recipes)
    
54. Standard saline citrate (SSC) (see Recipes)
    
55. NaCl-Tris-EDTA (NTE) buffer (see Recipes)
    
56. Tris-NaCl-Tween (TNT) buffer (see Recipes)
    
57. 20 mM iodoamide in NTE buffer (see Recipes)
    
58. DIG blocking solution (see Recipes)
    
59. 0.5% TSA blocking buffer (see Recipes)
    
60. Cy3-TSA amplification solution (see Recipes)
    

Equipment

1. Scissors
   
2. Surgical knife
   Note: We use set of stainless-steel mess handle (Feather, No.3) and stainless-steel spare blade (Feather, No.14).
   
3. Syringe
   
4. Syringe needle
   
5. Stereotaxic instrument (Narishige, model: SR-5M )
   
6. Surgical cotton
   
7. Pneumatic pump (Pneumatic Picopump) (World Precision Instruments, model: PV800 )
   
8. Puller (Narishige, model: PC-10 )
   
9. Glass pipette (Narishige, model: G-1.2 )
   
10. Peristaltic pump (ATTO Corporation, model: SJ-1211H )
    
11. Cork board
    
12. Microslicer (Leica Microsystems, model: VT1000S )
    
13. Razor blade
    
14. 10 ml tube
    
15. Confocal laser-scanning microscope (Olympus, model: FV1000 )
    
16. Hybridization oven (Bellco, model: 7930-00110 )
    
17. Water bath
    
18. Fluorescence microscope
    Note: Fluorescence signals of Alexa Fluor 488 and Cy3 or Alexa Fluor 594 are observed through fluorescence mirror units (Olympus, model: U-MWIBA3 , U-MWIG3 ).
    
19. Aspirator
    

Software

1. Confocal software (Olympus, FV10-ASW, ver.1.7)

Procedure