Visualization of Intracellular Tyrosinase Activity in vitro

Materials and Reagents

1. Glass coverslips (diameter-12 mm, No.1) (Polar Industrial Corporation, Blue Star, catalog number: 12mm Circular )
   Note: See Recipes for acid wash and sterilization.
   
2. Micro slides (L-75 mm x W-25 mm x h-1.35 mm) (Polar Industrial Corporation, Blue Star, catalog number: PIC-1 )
   
3. Plastic tissue culture (6 well) plate (Corning, catalog number: 3506 ) and bottle-top vacuum filter (pore size 0.22 μm) (Corning, catalog number: 430015 )
   
4. Melanocytes (Immortal wild type mouse melanocytes, melan-Ink4a-Arf-1 from C57BL/6J mice, referred to here as melan-Ink4a) [Resource: The Wellcome Trust Functional Genomics Cell Bank (Sviderskaya et al., 2010)]
   
5. Copper(II) sulphate pentahydrate (CuSO₄·5H₂O) (Sigma-Aldrich, catalog number: C7631 )
   
6. 3, 4-Dihydroxy-D-phenylalanine (D-DOPA) (Sigma-Aldrich, catalog number: D9378 )
   
7. 3, 4-Dihydroxy-L-phenylalanine (L-DOPA) (Sigma-Aldrich, catalog number: D9628 )
   
8. HCl (Sigma-Aldrich, catalog number: H1758 )
   
9. HNO₃ (Merck Millipore, catalog number: 101799 )
   
10. KCl (Sigma-Aldrich, catalog number: P9541 )
    
11. KH₂PO₄ (Merck Millipore, catalog number: 104873 )
    
12. NaCl (Fisher Scientific, catalog number: BP358-1 )
    
13. Na₂HPO₄·2H₂O (Fisher Scientific, catalog number: S472-500 )
    
14. Ethanol (70%) (Merck Millipore, catalog number: 818760 )
    
15. Fetal bovine serum (Biowest, catalog number: S1810-500 )
    
16. Formaldehyde (36.5-38% in H₂O) solution (HCHO solution) (Sigma-Aldrich, catalog number: F8775 )
    
17. Fluromount-G or mounting medium (SouthernBiotech, catalog number: 0100-01 )
    
18. Matrigel Matrix (Corning Matrigel Growth Factor Reduced Basement Membrane Matrix, Phenol Red-Free) (Corning, catalog number: 356231 )
    
19. Penicillin-Streptomycin (antibiotic) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
    
20. RPMI-1640 media (Thermo Fisher Scientific, Gibco^TM, catalog number: 31800-022 )
    
21. L-Glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030-081 )
    
22. 0.1% DOPA solution (see Recipes)
    
23. 4% Formaldehyde solution (see Recipes)
    
24. Growth media (see Recipes)
    
25. 0.1% Matrigel matrix solution (see Recipes)
    
26. 1x PBS (see Recipes)
    

Equipment

1. CO₂ incubator (maintained at 37 °C, 10% CO₂) (Thermo Fisher Scientific, model: Forma Water Jacketed CO₂ incubator )
   
2. Forceps (sterilized by autoclave)
   
3. Bright field microscope (Olympus Corporation, model: IX81 motorized inverted fluorescence microscope )
   
4. Hot-air-oven (Eyela, catalog number: NDO-420W )
   
5. Glass beaker (250 ml) (Borosil, catalog number: 1000D21 )
   

Procedure