Alexa fluor 568 or 488 goat anti mouse igg

Cells were fixed with 4% paraformaldehyde /4% sucrose for 30 min at room temperature and incubated for 1 h in blocking buffer (PBS containing 5% BSA, 5% normal goat serum, and 0.1% Triton X-100). The cells were then incubated with primary antibodies in blocking buffer at 4°C overnight. Primary antibodies included mouse monoclonal antibody against tyrosine hydroxylase (TH; 1:500; Sigma), rabbit polyclonal antibody against TH (1:50,000; Pel-Freez, Rogers, AR), rabbit polyclonal antibody against phospho-JNK (1:100; Cell Signaling), and rabbit polyclonal antibody against cleaved caspase 3 (1:1000; Cell Signaling). After three washes with PBS, the cells were incubated at room temperature for one hour with appropriate secondary antibodies: Alexa Fluor 488 (or 568) goat anti-rabbit IgG and Alexa Fluor 568 (or 488) goat anti-mouse IgG (1:200; Molecular Probes, Eugene, OR). Stained cells were monitored under a fluorescence microscope (Leica, Heidelberg, Germany).

Neuronal cultures were fixed with a solution of 4% paraformaldehyde and 4% sucrose, and blocked with a solution containing 5% BSA, 5% normal goat serum, and 0.1% Triton X-100 in PBS. Primary antibodies included mouse monoclonal antibody against tyrosine hydroxylase (TH; 1:500; Sigma), rabbit polyclonal antibody against TH (1:50,000; Pel-Freez, Rogers, AR), and rabbit polyclonal antibody against NeuN (1:2000; Sigma). Secondary antibodies were Alexa Fluor 488 (or 568) goat anti-rabbit IgG and Alexa Fluor 568 (or 488) goat anti-mouse IgG (1:200; Molecular Probes). Cells that immunostained positive for TH and had neurites that were twice the length of the soma were scored as TH⁺ neurons. Total TH⁺ neurons were counted on each culture base (an Aclar, a cover glass or a well of chamber slides).