Materials and Reagents

1. Mice
   
2. 0.9% sterile sodium chloride (NaCl) (Fresenius Kabi)
   
3. Ketamine hydrochloride (Ketavet, 100 mg/ml) (Pfizer)
   
4. Xylazine hydrochloride (Rompun, 20 mg/ml Xylazine) (Bayer)
   
5. 4% Paraformaldehyde in phosphate buffer (4% Roti-Histofix) (Roth, catalog number: P087.1 )
   
6. Hank’s balanced salt solution (HBSS) (Life Technologies, Invitrogen^TM, catalog number: 14170-138 )
   
7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
   
8. Sodium phosphate dibasic heptahydrate (Na₂HPO₄^.7H₂O) (Sigma-Aldrich, catalog number: S9390 )
   
9. Sodium phosphate monobasic monohydrate (NaH₂PO₄^.H₂O) (Roth, catalog number: K300.2 )
   
10. Potassium chloride (KCl) (AppliChem GmbH, catalog number: A3582 )
    
11. Potassium phosphate monobasic (KH₂PO₄) (Gerbu, catalog number: 2018 )
    
12. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
    
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. Chicken anti-GFP antibody (Aves, catalog number: GFP-1020 )
    
18. Goat anti-Doublecortin antibody (DCX, C18) (Santa Cruz, catalog number: sc-8066 )
    
19. Mouse anti-NeuN antibody (EMD Millipore, catalog number: MAB377 )
    
20. Donkey anti-chicken DyLight488 antibody (Dianova, catalog number: 703-485-155 )
    
21. Donkey anti-goat Alexa 647 antibody (Dianova, catalog number: 705-605-147 )
    
22. Donkey anti-mouse Alexa 546 antibody (Life Technologies, Invitrogen^TM, catalog number: A10036 )
    
23. Hoechst (33342) (Biotrend, catalog number: 40047 )
    
24. Gelatine to coat glass slides (Sigma-Aldrich, catalog number: G7041 )
    
25. Chromium (III) potassium sulfate dodecahydrate (Sigma-Aldrich, catalog number: 60152 )
    
26. Bromothymol Blue sodium salt (Sigma-Aldrich, catalog number: 114421 )
    
27. FD Rapid GolgiStain^TM Kit (FD NeuroTechnologies, catalog number: PK401 )
    
28. Millipore water
    
29. Ethanol (Sigma-Aldrich, catalog number: 459844 )
    
30. Xylene (Sigma-Aldrich, catalog number: 33817 )
    
31. Eukitt (Fluka, catalog number: 03989 )
    
32. Agarose (AppliChem GmbH, catalog number: A8963 )
    
33. Phosphate buffer saline (PBS) (20x) (see Recipes)
    
34. TBS (10x) (see Recipes)
    
35. TBS++ (see Recipes)
    
36. 0.1 M Phosphate buffer (see Recipes)
    
37. Gelatine to coat glass slides (see Recipes)
    
38. 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. Micro dissecting scissors
   
5. Forceps
   
6. Leica VT1200 Vibratome
   
7. Brush to transfer slices
   
8. Netwell carriers and plates (Corning Inc., catalog numbers: 3477 and 3520 )
   
9. Rocking platform
   
10. Tube roller mixer
    
11. Hot plate stirrer
    
12. Staining containers
    
13. Microscope glass slides
    
14. Cover slips
    
15. Confocal microscope
    
16. Centrifuge
    

Software

1. Amira Filament Editor Analysis (Visage Imaging) or other neuron morphology analysis software

Procedure