Axioskop

Serial coronal sections were examined at 10× magnification using a Zeiss Axioskop (Zeiss, Germany). Photographs were taken with an Olympus DP71 digital camera (resolution of 4140 pixels × 3086 pixels). Franklin and Paxinos (2008) stereotaxic coordinates were used to define specific brain regions: (i) the caudal pontine reticular nucleus (PnC), between -4.96 and -5.70 mm; (ii) the lateral globus pallidus (LGP), -0.10 and -1.06 mm; and the auditory cortex (AC), in the region between -2.18 and -3.64 mm. Both right and left hemispheres of three different sections separated by at least 140 μm for each area were examined. Within each section 3 regions of interest (ROI) were analyzed. The size of the three ROI was 200 × 200 μm. Using Image-pro 6.2.1 (Media Cybernetics) and ImageJ 1.50i (NIH), cells with threshold above background were counted. The researcher performing the analysis was blind to the experimental conditions. This procedure resulted in a total of 18 determinations of the number of cells stained with c-Fos within a specified area for each brain.

Approximately 1 g of each sample was homogenized and diluted in 1X PBS. Samples were filtered through a 0.2 μm pore size black polycarbonate membrane (Whatman International Ltd., Piscataway, NJ). Filtered cells were stained with 25 mg/mL acridine orange for 2 min in the dark. Unbound acridine orange was filtered through the membrane with 10 mL filter sterilized 1X PBS (Sigma Aldrich Corp., St. Louis, MI) and the rinsed membrane was mounted on a slide for microscopy. Cells were imaged with a FITC filter on a Zeiss Axioskop (Carl Zeiss, Inc., Germany).

Morphologic investigations were performed using a light microscope (Zeiss Axioskop; Carl Zeiss, Inc., Thornwood, New York, NY, USA) equipped with a device for differential contrast. Images were acquired using a digital camera NIKON COOLPIX 995. For each strain of the selected Scenedesmus, Tetradesmus and Desmodesmus genera grown at L₁₂₀ and L₂₀₀, with or without N-source, cell morphology was analyzed taking fifty measurements by ImageJ image analysis software [21 (link)]. Lipid droplet accumulation was also observed over the time in the strain of D. abundans grown under the different culture conditions. After Nile red staining (100 µg mL⁻¹) [22 (link)], cells were observed by confocal laser scanning microscope (CLSM; Olympus FV1000) using excitation at 488 and 635 nm and two channels for detecting chlorophyll a (650–750-nm emission range) and lipid labeled by Nile red (maximum emission = 572 nm). Three-dimensional images were constructed from series of 2D cross-sectional images (x-y plane) that were captured at 0.5-μm intervals along the z-axis using IMARIS 6.2.0 software (Bitplane AG, Zurich, Switzerland). The regions of interest (ROI) were evaluated in order to perform a spectral analysis in all the visible wavelength regions.

PS8568 animals expressing gfp in the XXX cells were induced to form dauers and then immobilized on a 4% agarose pad with 10 mg/ml levamisole. Laser ablation was performed on a Zeiss Axioskop (Carl Zeiss) equipped with an Andor MicroPoint nitrogen-pulsed laser microbeam (Oxford Instruments). XXX cells were visualized under fluorescence and the laser was fired at ∼5 Hz for a total of 20-30 pulses, or until all discernable fluorescence was gone. Cellular damage could often be visualized under DIC. Following surgery, animals were recovered onto a 35 mm NGM plate seeded with OP50 washed in S Basal and scored for dauer exit 24 h later. GFP was no longer discernable under stereomicroscopy in successfully ablated animals. Mock ablated animals were prepared and rescued identically to ablated animals but without receiving laser treatment.

Liver tissues were fixed with 4% paraformaldehyde and washed three times with PBS containing 0.1% Triton X-100. The tissues were embedded in O.C.T (Sakura, Japan) and the embedded block was sectioned using a Cryo-cut microtome (General Data Healthcare, Cincinnati, OH, USA) at 3–4 μm thickness. The sections were stained with the Oil Red O solution. The stained sections were observed under the light microscope (Zeiss Axioskop, Carl Zeiss, Inc., Jena, Germany). Staining area in tissues was evaluated using color-based threshold by Image J program (NIH, Bethesda, MD, USA). The total percentage of the staining area (lipid accumulation) was calculated as the sum of red-stained area divided by total area of microscopic field. Epididymal adipose tissues were fixed with 10% formalin and embedded in paraffin. The tissue blocks were sectioned at 8–10 μm and slide samples were obtained. The slides were stained with the H&E solution (Sigma, St. Louis, MO, USA) and observed under the light microscope. Adipocyte size was measured using Image J (NIH, Bethesda, MD, USA).

For light and electron microscopy, eyes were fixed at 4°C in 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). After washing in cacodylate buffer, the cornea and lens were removed and eye cups were hemisected. The halves were post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer and bloc-stained with uranyl acetate. The samples were dehydrated and embedded in Epon following standard procedures using reagents from AppliChem (Darmstadt, Germany), Merck (Darmstadt, Germany), and Serva (Heidelberg, Germany). Light microscopy on toluidine blue stained semi-thin sections (500 nm) was performed with a Zeiss Axioskop (Zeiss, Jena, Germany). Ultrathin sections (70 nm) were mounted on copper slot grids (Plano, Wetzlar, Germany) and stained with lead citrate and examined with a Zeiss 900 electron microscope (Zeiss, Jena, Germany).

The fixation and staining methods used for immunocytochemistry were performed as previously described (10 (link)). For immunostaining, AG1521 fibroblast cells were cultured on chamber slides and synchronized to the G0/G1 phases by contact inhibition. Following 30 min of treatment with monoglucosyl-rutin and radiation exposure at 37°C, the cells were fixed in 4% paraformaldehyde for 15 min, washed in PBS and then treated with 0.5% Triton X-100 for a further 10 min. Non-specific binding sites were inhibited using PBS with 10% goat serum at 4°C overnight. The chamber slides were then incubated with mouse monoclonal anti-γ-H2AX antibody (Millipore, Billerica, MA, USA) for 1 h, washed three times in PBS and incubated with Alexa 488 conjugated goat anti-mouse secondary antibody (Invitrogen Life Technologies) for 1 h at 37°C. The cells were washed four times in PBS and the nuclei were stained with Antifade Gold with DAPI solution (Invitrogen Life Technologies). Images of the cells were captured using a Z-stage motorized Zeiss Axioskop with Metamorph system (Carl Zeiss AG, Jena, Germany). The z-stacked images were stored and 50 cells from these were later scored in three independent experiments.