Eclipse ni epifluorescence microscope

On day 7 of growth, two samples (10 mL) containing the highest concentrations of each antibiotic were centrifuged at 3,500 rpm for 10 min. The algal cells obtained from one sample were fixed with 2.5% glutaraldehyde and treated as per the modified method described by Lapaille et al.^{68 (link)} for microstructure analysis using transmission electron microscopy (TEM, HT7700, Hitachi, Japan).
The other sample was used for the detection of DNA damage with single cell gel electrophoresis or comet assay. The protocol used was as per the method described by Tice et al.⁶⁹ with some modifications. Images were digitised using light and fluorescence microscopy with an Eclipse Ni epifluorescence microscope (Nikon) equipped with an inbuilt white and external mercury lamp light source (Model C-SHG1; Nikon). For documentation, a DS-Ri2 digital sight camera (Nikon) was used. Fifty nucleoids were evaluated from each group.

Cell viability was assessed through culture-independent methods using a live/dead stain. Briefly, cells were stained on black 0.22 µm polycarbonate Isopore filters using equal volumes of SYTO 9 and propidium iodide. Stained cells were then viewed using a Nikon ECLIPSE Ni epifluorescence microscope in sixty random fields of view. Cells that stained fluorescently green (and not red) were considered intact and viewed cells were inferred to be viable. All cells (viable or not) were quantified in relation to the total volume of air sampled.

To determine the subcellular localization of lamin A/C and prelamin A, OS cells were seeded in 12-well plates and the cells were grown on coverslips, fixed with methanol for 8 min at −20 °C. Cells were then blocked with 3% BSA-containing PBS for 1 h. Antibodies diluted in 3% BSA-containing PBS were applied overnight at 4 °C, and revealed by using FITC-conjugated secondary antibodies diluted 1:200 (incubated for 1 h at RT). Samples were mounted with a DAPI-containing anti-fade reagent (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) and observed with a Nikon Eclipse Ni epifluorescence microscope. The images captured with NIS-Elements 4.3 AR software were processed using Photoshop CS4 (Adobe Systems, Inc., San Jose, CA, USA). The following antibodies were used: anti-prelamin A (Merck Millipore, Burlington, MASS, USA), diluted 1:500, anti-lamin A/C (#4777 Cell Signaling Tecnhology, Danvers, MA, USA), diluted 1:100.

Cells grown on coverslips were fixed with absolute methanol at −20°C for 7 min. After saturation with 4% BSA in PBS, coverslips were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 hr at RT. Samples mounted with antifade reagent were observed with a Nikon Eclipse Ni epifluorescence microscope. Images captured with NIS‐Elements 4.3 AR software were elaborated using Photoshop CS.

Images were taken on a Nikon Eclipse-Ni epifluorescence microscope coupled to a Nikon Digital Sight DS-U3 camera, where images were acquired using Nis-Elements (4.10.04). Adobe Photoshop v8.0 (Adobe Systems, Mountain View, CA, USA) was used to process for brightness and contrast. To assess the GFAP immunoreactivity, three coronal sections from each brain covering 160 µm were assessed within the area where the sensor tip had been placed. Three regions of interest (ROI) (144 × 144 µm) were selected in each section using ImageJ software (http://rsb.info.nih.gov/ij/). The average intensity was assessed for each ROI, and the mean for that section calculated. For the corpus callosum, ROI was taken at the left, centre and right of the section. The superficial layers of the cortices were assessed, with ROI selected to the left, centre and right of the section. All images were acquired at the same imaging session with the same microscope settings throughout.