Dc300 camera

The models were fixed in 1% paraformaldehyde (PFA)/PBS and rinsed three times in PBS. Cell morphology was assessed by adding 4% crystal violet (BD Biosciences) to the cells. After 5 minutes of staining, the cells were rinsed and imaged using light microscopy at 100x magnification (Leica DMIL microscope and Leica DC300 camera). Morphology was quantified using a previously developed protocol using Matlab^TM software [26 (link)]. Briefly, the shape index (SI) is a previously defined parameter used to characterize the degree of cell elongation where the SI of a circle is equal to 1 and a straight line is equal to 0.

Twenty larvae were sampled each day from each tank to track changes in length, weight, and biochemical condition. After removing larvae from the tank, they were photographed (WILD M8 stereomicroscope, Olympus SZH and a Leica DC 300 camera, using Motic Images 2.0 software), dipped in de-ionized distilled water, and individually frozen at -80°C. Measurements of SL and yolk sac area were made using image analysis software (Image J, version 1.43u, freeware, Wayne Rasband, NIH, USA). All frozen larvae were freeze-dried (Christ Alpha 1–4 LSC, 0.200 mbar; >16 h) and weighed (Sartorius Genius SE2 microbalance, DW ± 0.1 μg).
In order to have a sufficient amount of tissue to measure nucleic acids, 2 or 3 freeze-dried larvae were combined and the tissue was homogenized with 1% sarcosil Tris-EDTA buffer (Sigma-Aldrich, Hamburg, Germany) and glass beads (0.2–2.1 mm) in a Retsch shaking mill (both Retsch, Haan, Germany). Following a modified protocol from Caldarone [33 ], amounts of RNA and DNA were determined spectrofluorometrically (using the aforementioned equipment) with ethidium bromide as a fluorescence-dye and restriction enzymes to eliminate the nucleic acids (as in [34 ]). The ratio of RNA to DNA was standardized (sRD) using methods outlined by Caldarone [35 ] using a factor of 2.4.

Toluidine blue-stained 1-μm semi-thin cross sections of optic and sciatic nerves were observed under an upright Zeiss Axiophot microscope and images were captured using a Leica DC300 camera. For the optic nerve, the numbers of myelinated axons were counted per field in four randomly sampled fields per section, using ImageJ, and the total number of fibers was calculated for the total area of the cross section. For the sciatic nerve, the total numbers of myelinated axons or Remak-bundles of non-myelinated axons were counted per cross-section, using ImageJ. The diameters of myelinated axons and axons plus myelin were calculated by hand-tracing and measuring the corresponding circumferences, using ImageJ. The g-ratio as an index of myelination, was calculated for each fiber by dividing the inner diameter of the axon to the total diameter of the myelinated fiber. A minimum of 100 axons were measured in each nerve and values were averaged from 3–4 nerves per genotype.

Five-day-old seedlings grown on half-strength MS agar plates were imaged using Leica MZ9.5 stereomicroscope coupled to Leica DC300 camera. For hair density of rop mutants, the number of hairs on the first 4 mm from the tip were manually counted on ten growing roots for each genotype in three independent biological replicates (n=30). For the yip4 mutants and complementation lines, the number of hairs was counted from 5 to 10 mm away from the tip (n=20 seedlings from two biological replicates). Lengths from all hairs present on the first 4 mm from the tip were measured using ImageJ on ten growing roots for each genotype in three independent biological replicates with minimum 150 hairs measured per genotype (n=450). A Student's t-test was used to check for statistical difference (two-tailed with equal variance).

One statolith per medusa was used for analyses as it was determined one statolith is representative of the other three in a medusa [42 (link)]. A section of black electrical tape on a glass slide had a minute amount of glue smeared onto it which allowed for temporary adhesion. The statolith was placed in this temporary adhesion orientated proximal face up (cleavage vertical; see Fig 1). The statolith was illuminated using a cold lamp source then photographed and measured using Leica IM50 software (Version 4.0 Release 132, Leica Microsystems Imaging Solutions Ltd. 2004) coupled with a Leica DC300 camera fitted to a Leica DMLB microscope. The same procedure was then followed taking images of the statolith oral (cleavage down) and lateral (cleavage horizontal) faces (Fig 1). Greyscale statolith images including a 100 μm scale bar were then inverted and some minor editing done (removal of statocyst membrane fragments that were not sulphate material, or local blurring, after consulting original image) using Adobe Photoshop CS5.1 leaving a black statolith silhouette on white background in Tagged Image File Format (TIFF).

Adhesion tests were performed in 6-well plates (Life Sciences, BD Falcon, Tewksbury, MA, USA). Cells (1 × 10⁶) were inoculated into wells and incubated at 37 °C in an environment containing 5% CO₂ for 16 h. Subsequently, cells in suspension were collected in a 15 mL conical tube, centrifuged at 1000 rpm for 5 min at room temperature, resuspended in a specific volume of culture medium, and counted using the Countess Automated Cell Counter (Invitrogen, Thermo Fisher Scientific). The adhered cells were trypsinized and counted. Senescence-associated (SA) β-galactosidase activity was detected as described elsewhere [27 (link)]. Briefly, senescence tests were carried out 7 d after puromycin selection of lentiviral transduced cells. Adhered cells were rinsed twice in PBS 1X and fixed in 2% formaldehyde-0.2% glutaraldehyde for 5 min at room temperature. After two washes in PBS 1X, cells were incubated in freshly prepared staining solution (40 mM citric acid/sodium buffer phosphate, 5 mM K₄ [Fe (CN)₆], 5 mM K₃ [Fe (CN)₆], 150 mM NaCl, 2 mM MgCl₂, 1 mg/mL X-Gal, pH 6.0) at 37 °C for 3 h (Caco-2/15) or 8 h (HT-29). Cells were washed twice in PBS 1X and then methanol before letting them air dry. The plates were photographed using a DMIL microscope equipped with a Leica DC300 camera (Leica Camera, Allendale, NJ, USA).