Alexa fluor 488 labeled goat anti rabbit secondary antibody

Alexa Fluor 488-labeled goat anti-rabbit secondary antibody, 5 mM Sytox orange and 4′,6-diamidino-2-phenylindole (DAPI) were all purchased from Life Technologies (Pty) Ltd (USA). Histopaque-1077 was purchased from Sigma Aldrich (Pty) Ltd (Johannesburg, South Africa) and the polyclonal rabbit anti-histone H4 (citrulline 3) was purchased from Merck Millipore (Pty) Ltd (Eastern Cape, South Africa). Propidium iodide (50 µg/ml DNA Prep-Stain) was purchased from Beckman Coulter Pty (Ltd) (Johannesburg, South Africa) and the cigarette smoke condensate (CSC) from Murty Pharmaceuticals Inc, Lexington (KY) and dissolved in dimethyl sulphoxide to give a stock concentration of 40mg/ml. The total amount of condensate generated during the combustion of one cigarette is about 26.3 mg, therefore the concentrations of the condensate used in this study were relevant in the context of the smoking habit21 (link). The CSC was prepared by burning University of Kentucky's 1R3E standard cigarettes and extracting the total particulate matter generated into DMSO using a smoking machine 22 (link)–23 (link). The constituents of CSC include a complex mixture of phenolic compounds such as phenols, cresols and dihydroxybenzenes, as well as hydrogen cyanide, acrolein and formaldehyde24 –25 (link). Unless indicated the remaining chemicals and reagents mentioned in the methods were purchased from Sigma-Aldrich.

To examine the intensity and the pattern of LC3-II immunostaining, the cells were seeded on glass cover slides in 12-well plates and left to attach overnight. After the treatment with fisetin and/or paclitaxel, the cells were fixed with 4 % paraformaldehyde (Serva; Heidelberg, Germany) in PBS for 30 min, washed three times with PBS, permeabilized with 0.25 % Triton X-100 (Serva; Heidelberg, Germany) in PBS for 5 min and blocked with 1 % BSA (Sigma-Aldrich; St. Louis, MO, USA) in PBS (BSA–PBS pH 7.6) for 20 min. The staining of LC3-II was performed using the rabbit anti-LC3-II antibody (Thermo Scientific; Rockford, USA) diluted 1:2000 in 1 % BSA–PBS (1 h, RT, a moist chamber). After rinsing three times with 1 % BSA–PBS, the cells were incubated with Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (Life Technologies Corp.; Carlsbad, CA, USA) diluted 1:100 in PBS (60 min, RT, a moist chamber in the dark). Following three washing steps with PBS, the cell nuclei were counterstained with DAPI (diluted 1:20,000 in distilled water; Sigma-Aldrich; St. Louis, MO, USA) for 10 min. Finally, the slides were rinsed three times with distilled water, mounted with Aqua-Poly/Mount (Polysciences; Warrington, PA) and examined using Nikon Eclipse E800 fluorescence microscope and NIS-Elements 4.0 software (all from Nikon; Tokyo, Japan).

Three-to-five-day-old adult male flies that were fed with or without 1% sodium chloride in sucrose/agar food for 3 days were collected for brain dissection at ZT2. Brains were fixed in 4% buffered formaldehyde at 25°C for 2 h, washed in PBS (pH 7.4) with 0.2% Triton X-100 (PBT), blocked in 5% goat serum in PBT (PBST) for 30 min at room temperature, and incubated in rabbit anti-TH (Millipore, #AB152) overnight at 4°C. After three 15 min washes with PBT, the brains were incubated with Alexa Fluor^® 488-labeled goat anti-rabbit secondary antibody (Life Technologies) overnight at 4°C, rinsed thoroughly and mounted. Images were taken under confocal microscopy (Leica TCS SP5) at an optical section thickness of 1–2 μm and were analyzed with Image J (NIH).