Materials and Reagents

1. Filter paper, 55 mm (Sigma-Aldrich, Whatman^®, catalog number: WHA1441055 )
   
2. Millicell organotypic filter inserts 0.4 µm, 30 mm (EDM Millipore, catalog number: Picmorg50 )
   
3. 6-well culture dishes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 140675 )
   
4. 24-well histology trays (Sigma-Aldrich, CELLSTAR^®, catalog number: 662160 )
   
5. 50 mm sterilin Petri dish (Camlab, catalog number: p17118 )
   Note: This link leads to a range of Petri dishes.
   
6. Pipette tips, 1,000 µl (SARSTEDT, catalog number: 70.762.010 )
   
7. Pipette tips, 200 µl (SARSTEDT, catalog number: 70.760.012 )
   
8. Syringe
   
9. Glass microscope slides (Academy Science, catalog number: N/A143 )
   
10. Glass coverslips (VWR, catalog number: 631-0133 )
    
11. Tin foil
    
12. Mice (8-20 days old)
    
13. Ethanol (Sigma-Aldrich, catalog number: 32221 )
    
14. Acetone (Alfa Aesar, catalog number: L10407 )
    
15. Sodium pentobarbitone (Pentoject) (Animalcare, catalog number: XVD-132 )
    
16. 5-ethynyl-2’-deoxyuridine (EdU) (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: A10044 )
    
17. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
    
18. Phosphate buffered saline (Dulbecco A) (PBS) (Oxoid Limited, catalog number: BR0014 )
    
19. Triton X-100 (VWR, catalog number: 28817.295 )
    
20. Copper sulphate pentahydrate (CuSO₄·5H₂O) (VWR, catalog number: 84845.230 )
    
21. Biotin-azide (Kerafast, catalog number: EVU101 )
    
22. Ascorbic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12950364 )
    
23. Streptavidin Alexa⁵⁵⁵ conjugate (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: S-32355 )
    
24. Vectashield mounting medium (Vecta Laboratories, catalog number: H-1000 and H-1200 )
    
25. DAPI (4’,6-diamidino-2-phenylindole)
    
26. Nail lacquer
    
27. Anti-PKD2L-1 (polycystic kidney disease 2-like 1 protein)
    
28. PBS tablets
    
29. Sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10667823 )
    
30. Sodium phosphate dibasic anhydrous (Na₂HPO₄) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10396313 )
    
31. Trizma base (Sigma-Aldrich, catalog number: T1503 )
    
32. Trizma hydrochloride (Sigma-Aldrich, catalog number: T3253 )
    
33. Sucrose (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10386100 )
    
34. Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: 31437 )
    
35. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 746436 )
    
36. Magnesium sulphate (MgSO₄·7H₂O) (VWR, catalog number: 25165.260 )
    
37. Glucose (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10539380 )
    
38. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: 21114 )
    
39. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
    
40. Penicillin and streptomycin (Sigma-Aldrich, catalog number: P4458 )
    
41. Neurobasal A medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 10888-022 )
    
42. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
    
43. B-27 supplement (Thermo Fisher Scientific, Gibco^TM, catalog number: 17504-044 )
    
44. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F6178 )
    
45. Phosphate buffered saline (PBS) (see Recipes)
    
46. 0.2 M phosphate buffer (PB) (see Recipes)
    
47. 0.1 M Tris buffer (see Recipes)
    
48. Sucrose artificial cerebrospinal fluid (see Recipes)
    
49. 8% paraformaldehyde solution (PFA) (see Recipes)
    
50. 4% paraformaldehyde solution in 0.1 M PB (see Recipes)
    
51. Dissection medium (see Recipes)
    
52. Culture medium with serum (see Recipes)
    
53. Culture medium serum free (see Recipes)
    

Equipment

1. Autoclave (Dixons Surgical Instrument, model: VARIO 1528 )
   
2. P1000 pipetman classic pipette (Gilson Scientific, catalog number: F123602 )
   
3. P20 pipetman classic pipette (Gilson Scientific, catalog number: F123600 )
   
4. Laminar flow hood (biological safety cabinet) (The Baker Company, model: Steril Gard class II type A )
   
5. CO₂ incubator (Panasonic, model: MCO-18AC-PE )
   
6. Dissection microscope (Vickers, instruments)
   
7. Dissection equipment (Figure 1)
1. Dissection scissors
   
2. Spring scissors
   
3. Fine forceps x 2
   
4. Microspatula
   
8. Tissue chopper (McIlwain)
   
9. Razor blades (Wilkinson sword)
   
10. Shaker plate (IKA, model: Vibrax-vxr )
    
11. Fine paint brush
    
    
    Figure 1. Equipment used for dissection
    

Procedure