Immunocytochemical analysis was used to establish the presence of cleaved p65-NFκB in TH positive dDCNs following 6OHDA treatment. dDCNs were grown and exposed to 100 µm 6OHDA for 2 h as described above. On the next day, control and 6OHDA-treated dDCNs were fixed with 4% paraformaldehyde for 15 min and were washed with cold PBS. Subsequently, cells were treated with 0.1% Triton X-100 (10 min) and blocked with 10% goat serum for 40 min prior to incubation with the primary antibody (anti-TH, 1:1500,) and with cleaved (p65 subunit) anti-NFκB (1:1500) overnight at 4oC, and incubated with the secondary antibody (sheep anti-rabbit IgG-FITC, 1:800) (sheep-anti-mouse IgG rhodamine, 1:300) for 2 h at room temperature. dDCNs were mounted using Vectashield mounting medium and viewed under a Meiji fluorescent microscope (Mazurek, Warwickshire, UK). Furthermore, co-localisation studies were performed to determine if NFκB and TH (see Figure 1), and NFκB and caspases (see Figure 2) were present in the same cell by using the primary antibodies anti-TH (1:1500), anti-NFκB, p65 subunit (1:1500), anti-caspase-3 (1:1000), anti-caspase-2 (1:2000) and anti-caspase-8 (1:2000), and the secondary antibodies donkey anti-mouse IgG-FITC (1:500) and goat anti-rabbit IgG-rhodamine (1:2500). Control and treated dDCNs were counted under × 20 magnification in 5 fields of vision per area (1.428 mm × 1.092 mm). To analyse co-localisation data, cells localised in the same field and stained with two different fluorochromes were counted. Cell numbers are expressed as the mean per selected field from the wells. Three independent experiments were performed for each sample prior to statistical analysis (Student’s t-test, p < 0.05), as shown in Figure 1B and Figure 2B.
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