Immunofluorescence Labeling of Tyrosine Hydroxylase and Dopamine Beta-Hydroxylase

This method was similar to previous work from our laboratory (Fan et al., 2011 (link)). Briefly, the slides containing the sections of LC regions were pre-incubated in 5% bovine serum albumin in phosphate-buffer saline (PBS) supplemented with 0.2% Triton-X 100. These slides were further exposed to primary monoclonal antibody solution (for DBH: 1:2000, ab19353, RRID: AB_73185, Abcam, Cambridge, MA, USA; for TH: 1:1000, MA1-24654, RRID: AB_79566, ThermoFisher Scientific, Waltham, MA, USA) overnight at 4°C. After washing, slides were then probed with 2nd antibodies (for DBH: Alexa Fluor 488-conjugated goat anti-rabbit IgG; for TH: Alexa Fluor 488-conjugated Goat anti-mouse IgG; both from Abcam, Cambridge, MA, USA), followed by 4 time rinses with 0.1 M PBS. A Leica TCS SP2 confocal microscope system (Leica Microsystems Inc., Bannockburn, IL, USA) was used to observe and acquire immunofluorescence labeling. These images were quantitatively quantified using ImageJ software (Rasband, US National Institutes of Health, Bethesda, http://rsbweb.nih.gov/ij, 2010). Non-immunoreactive portions of brain sections adjacent to the LC region were taken as the reference background levels.