Te2000 inverted microscope

The production of superoxide in myocardial tissue was detected using dihydroethidium (DHE) staining under a fluorescence microscope. Briefly, myocardial tissue sections (5 μm) were prepared and then incubated (30 min, 37 °C) in Krebs-HEPES buffer (mM components: NaCl 99, KCl 4.7, MgSO₄ 1.2, KH₂ PO₄ 1.0, CaCl₂ 1.9, NaHCO₃ 25, glucose 11.1, Na HEPES 20; pH 7.4) containing 2 μM DHE in a dark room. The slides were examined with a Nikon TE2000 inverted microscope (Nikon, Tokyo, Japan) at excitation and emission wavelengths of 480 and 610 nm, respectively.
The thiobarbituric acid method (Beyotime) was used to detect the level of malondialdehyde (MDA) in the myocardium. The total antioxidant capacity (T-AOC) of the myocardium was measured using the T-AOC detection kit using the plasma iron reduction capacity method (Beyotime). The activities of total SOD, Cu-Zn/SOD, and Mn-SOD in the myocardium were determined using the WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-)-2 H-tetrazole) method (Beyotime).

Paraffin-embedded tumor samples were sectioned (4 μm) and mounted on microscopic slides. Heat-induced epitope retrieval was performed in 10 mmol/L citric acid buffer at pH 7.2 preheated in a microwave and the sections were heated at 98 °C for 20 min. Sections were incubated with the primary antibody Ki67 (DAKO M7240, clone MIB-1, 1:250) at 4 °C overnight, rinsed with TBST, and incubated for 1 h at room temperature with biotin-conjugated secondary antibody, followed by 30 min incubation with ABC complex (VECTASTAIN^® ABC Kit, Vector Laboratories, Burlingame, CA, USA). Visualization was achieved using DAB chromogen (DAKO K3468) as the substrate, and slides were counterstained with haematoxylin (CellPath Ltd., Newtown, UK) and mounted with Entellan^® new (Merck Millipore, Burlington, MA, USA, 107961). Images were obtained using a Nikon TE2000 inverted microscope (Nikon Instruments Inc., Melville, NY, USA), and the percentages of Ki-67 positive cells were calculated, counting positive and negative cells using the point tool in the ImageJ freeware.

Intracellular reactive oxygen species (ROS) were assessed using the cell membrane permeable probe dihydro‐rhodamine‐123 (DHR‐123) (#D23806; ThermoFisher Scientific, Waltham, MA, USA) according to a procedure described before (Rousseau et al. 2013). Measurements were made at a stage where ROS production is optimal in cultures exposed to PDC, that is 24 h after initiation of the treatment. For each culture condition, fluorescent images of 6 randomly chosen fields were acquired with a 20× fluorescence objective using a Nikon TE 2000 inverted microscope (Nikon, Champigny‐sur‐Marne, France) equipped with an ORCA‐ER digital camera and the HCimage Imaging software (Hamamatsu, Corp., Bridgewater, NJ, USA). Results were expressed in fractional change in fluorescence intensity relative to baseline in control cultures (F/F₀) as described previously for other fluorescent probes (Toulorge et al. 2011; Sepulveda‐Diaz et al. 2016). The open source ImageJ software (Rasband 1997–2016) was used for quantification of the fluorescent signals. Note that because of the technical constraints imposed by our model system, we extrapolated ROS data from the whole population of neuronal cells to the few DA neurons present in midbrain cultures.