Materials and Reagents

1. Ketamine hydrochloride (100 mg/ml) (Ketaset FORT DODGE)
   
2. Xylazine hydrochloride (100 mg/ml) (Xylazine AnaSed)
   
3. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
   
4. Sodium Phosphate monohydrate (NaH₂PO₄^.1H₂O) (Sigma-Aldrich, catalog number: S9638 )
   
5. Sodium Phosphate heptahydrate (NaH₂PO_4^.7H₂O) (Sigma-Aldrich, catalog number: S9390 )
   
6. Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359 )
   
7. Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
   
8. Phosphate buffer (PB)
   
9. Phosphate buffer saline (PBS)
   
10. EDTA (Sigma-Aldrich, catalog number: E7889 )
    
11. Blocking buffer
    
12. Normal goat serum (NGS) (Sigma-Aldrich, catalog number: G-6767 )
    
13. Saponin (Sigma-Aldrich, catalog number: 47036 )
    
14. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
    
15. Primary Antibodies
    
    1. Rabbit anti-myosin VIIa antibody (a hair cell-specific marker) (Proteus Biosciences, catalog number: 25-6790 ) at a dilution of 1:50 in blocking buffer.
       
    2. Guinea pig anti- VGLUT3 (Vesicle Glutamate Transporter 3) antibody at 1:5,000 in blocking buffer (a gift from Dr. Robert Edward, Department of Neurology, UCSF)
       
16. Secondary Antibodies
    1. Cy2-conjugated Goat anti-rabbit secondary IgG antibody (Jackson ImmunoResearch) at a dilution of 1:4,000 in PBS.
       
    2. Cy3-conjugated Goat anti-guinea pig secondary IgG antibody (Jackson ImmunoResearch) at a dilution of 1:4,000 in PBS.
       Note: secondary antibodies should be diluted in PBS (not blocking buffer).
17. DAPI to counterstain nuclei (1.5 μg/ml in PBS) (Sigma-Aldrich, catalog number: D9564 ).
    
18. Antifade FuorSave reagent (Calbiochem, catalog number: 34589 )
19. 10x PB (see Recipes)
20. 10x PBS (see Recipes)
21. 4% PFA-PB (50 ml) (see Recipes)
22. Blocking buffer (see Recipes)
    

Equipment

1. Syringe (1 ml syringe 25G1) (BD Bioscience, catalog number: 329622 )
   
2. 0.5 ml microcentrifuge tube’s caps (Thermo Fisher Scientific, catalog number: 02-681-311 )
   
3. Microdissecting scissor (Fine Science Tools, catalog number: 15002-08 )
   
4. Fine forceps (Ted Pella, catalog number: 5621 )
   
5. Surgical blade (Feather)
   
6. 5 ml Vials (Denville, catalog number: T8200-S )
   
7. Pasteur pipettes (used to rinse the tissue) (Thermo Fisher Scientific, catalog number: 13-678-20 )
   
8. SuperFrost slides (Thermo Fisher Scientific, catalog number: 12-550 )
   
9. Cover slips (Thermo Fisher Scientific, catalog number: 12-548-5A )
   
10. Petri dish (BD Biosciences, Falcon^®, catalog number: 1007 )
    
11. Aluminum foil
    
12. Refrigerator
    
13. Rocking platform
    
14. Dissecting microscope
15. Confocal microscope

Procedure