Materials and Reagents

1. Mice
   
2. 5-Bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich, catalog number: B5002 )
   
3. 0.9% sterile sodium chloride (NaCl) (Fresenius Kabi)
   
4. Ketamine hydrochloride (Ketavet, 100 mg/ml) (Pfizer)
   
5. Xylazine hydrochloride (Rompun, 20 mg/ml Xylazine) (Bayer)
   
6. 4% Paraformaldehyde in phosphate buffer (4% Roti-Histofix) (Roth, catalog number: P087.1 )
   
7. Hank’s balanced salt solution (HBSS) (Life Technologies, Invitrogen^TM, catalog number: 14170-138 )
   
8. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
   
9. Sodium phosphate dibasic heptahydrate (Na₂HPO₄^.7H₂O) (Sigma-Aldrich, catalog number: S9390 )
   
10. Sodium phosphate monobasic monohydrate (NaH₂PO₄^.H₂O) (Roth, catalog number: K300.2 )
    
11. Potassium chloride (KCl) (AppliChem GmbH, catalog number: A3582 )
    
12. Potassium phosphate monobasic (KH₂PO₄) (GERBU Biotechnik GmbH, catalog number: 2018 )
    
13. Hydrochloric acid (HCl, 37%) (Sigma-Aldrich, catalog number: 30721 )
    
14. Trizma base (Sigma-Aldrich, catalog number: T1503 )
    
15. Horse serum (Biochrom, catalog number: S9135 )
    
16. Triton X-100 (Sigma-Aldrich, catalog number: X-100 )
    
17. Boric acid (Fluka, catalog number: 15660 )
    
18. Sodium tetraborate decahydrate (Sigma-Aldrich, catalog number: S9640 )
    
19. Hoechst (33342) (dilute with H₂O to 10 mg/ml) (Biotrend, catalog number: 40047 )
    
20. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
    
21. Superglue
    
22. Antibodies (see Table 1 and 2)
    
23. Agarose (AppliChem GmbH, catalog number: A8963 )
    
24. Phosphate buffered saline (PBS) (20x) (see Recipes)
    
25. TBS (10x) (see Recipes)
    
26. TBS++ (see Recipes)
    
27. Boric buffer (see Recipes)
    
28. 0.1 M Phosphate buffer (PB) (see Recipes)
    
29. Mowiol (Merck/Calbiochem, catalog number: 475904 ) (see Recipes)

Equipment

1. Syringe (1 ml syringe 27 G for i.p. injections)
   
2. 0.5 ml Eppendorf Safelock tubes
   
3. 15 ml and 50 ml Falcon tubes
   
4. Petri dish
   
5. Micro dissecting scissors
   
6. Forceps
   
7. Leica VT1200 Vibratome
   
8. Brush to transfer slices
   
9. Netwell carriers and plates (Corning Incorporated, catalog number: 3477 and 3520 )
   
10. Rocking platform
    
11. Tube roller mixer
    
12. Microscope glass slides
    
13. Cover slips
    
14. Confocal microscope
    
15. Shaker
    
16. Centrifuges
    

Software

1. ImageJ

Procedure