Cells on chamber slides were fixed with 4 % paraformaldehyde (Nakarai, Kyoto, Japan) for 20 min, washed with 10 mM phosphate-buffered saline (PBS, pH 7.4), and blocked with 2.5 % normal goat serum (Vector Laboratories, Burlingame, CA) for 20 min at 23 °C. Cells were then incubated with rabbit anti-tyrosine hydroxylase (TH) antibody (1:1000 dilution, Protos Biotech Corporation, New York, NY) diluted in 10 mM PBS containing 0.1 % Triton X-100 (0.1 % PBST) for 18 h at 4 °C. The secondary antibody was goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200 dilution, Molecular Probes, Eugene, OR). The cells were counterstained with Hoechst nuclear stain (10 µg/ml, Molecular Probes) for 2 min and washed before mounting with Fluoromounting medium (Dako Cytomation, Glostrup, Denmark).
All slides were observed under a fluorescence microscope (Olympus BX50-FLA, Tokyo, Japan) using a mercury lamp using a 470–490 nm or a 360–370 nm band-pass filter to excite Alexa Fluor 488 or Hoechst dye, respectively. Light emitted from Alexa Fluor 488 or Hoechst dye was collected through a 515–550 nm band-pass filter or a 420 nm long-pass filter, respectively. Adobe Photoshop CS4 software (Adobe, Waltham, MA) was used for digital amplification of the images.
All slides were observed under a fluorescence microscope (Olympus BX50-FLA, Tokyo, Japan) using a mercury lamp using a 470–490 nm or a 360–370 nm band-pass filter to excite Alexa Fluor 488 or Hoechst dye, respectively. Light emitted from Alexa Fluor 488 or Hoechst dye was collected through a 515–550 nm band-pass filter or a 420 nm long-pass filter, respectively. Adobe Photoshop CS4 software (Adobe, Waltham, MA) was used for digital amplification of the images.