Irdye 800cw anti rabbit antibody

Protein concentrations were examined using a Piece BCA protein assay kit (Thermo Fisher Scientific). A total of 10µg of the protein of each sample was loaded in a lane of the precast NUPAGE 4–12% Bis-Tris gels (Thermo Fisher Scientific). Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane for two hours at 400 mA. The membrane was blocked for 30 min using an Intercept blocking buffer (LI-COR) and the primary antibodies against TH (1:2500, Abcam, Cambridge, UK) or GAPDH (1:10,000, Millipore, Burlington, MA, USA) were applied and incubated overnight at 4 °C. After being washed with 1x PBST (0.1% Tween 20 in PBS) 4 times, the membrane was incubated with the fluorescent conjugated secondary antibodies (IRDye680RD anti-mouse antibody [1:20,000] or IRDye800CW anti-rabbit antibody [1:30,000], LI-COR, Lincoln, NE, USA) for one hour at room temperature. Membranes were scanned using an Odyssey Fc Imaging system (Millennium Science, Mulgrave, VIC, Australia). The intensity of specific bands for TH or GAPDH and their relative expression were quantified using Odyssey software.