Dp71 microscope digital camera

Cells were serum starved in medium with 0.5% FCS for 24 h while sub-confluent, then harvested, resuspended in serum deficient medium, and seeded into 8 μm Transwell inserts at 1.25 × 10⁴ cells per insert and placed into 24-well companion plates containing DMEM + 10% FCS as a chemoattractant stimulus. After 4 h cells were fixed with 4% paraformaldehyde, stained with 3% crystal violet, and non-migratory cells removed from the inside of the insert with a cotton bud. Membranes were then imaged using an inverted research microscope (Olympus, Tokyo, Japan) with DP71 microscope digital camera (Olympus, Tokyo, Japan) with six images taken per membrane. Each set of images was taken in the same place for each membrane to minimize any bias and ensure consistency. Images were then analysed using ImageJ (Image Processing and Analysis in Java; US National Institutes of Health, Bethesda, MD, USA). Data are presented as the number of cells per field, which represents the mean of the counts across the six fields for each membrane. Replicate measurements (number of cells per field) were then combined and data analysed using unpaired t-tests.

High vein density lines from the mutant populations were subjected to detailed microscopy analysis. Vein counts per 2 mm and cellular arrangements were viewed with a light microscope (Olympus BX51 Motorized Research Microscope, Tokyo, Japan, connected to an Olympus DP71 Microscope Digital Camera, Tokyo, Japan). Images were taken using Olympus CellˆP imaging software (from the Olympus Cell^∗ software family) and had their parameters quantified using ImageJ (Version 1.44).
Vein density viewings were taken of fresh leaf samples at 4x magnification. Samples were then fixed in FAA (formalin-acetic acid-alcohol solution) (500 ml absolute ethanol : 270 ml formaldehyde : 50 ml acetic acid : 180 ml water) for at least 12 h. These were cross-sectioned by hand using a razor blade. Leaf sections were placed in 85% ethanol for 24 h, after which the ethanol was removed and rinsed off with water. A fresh application of 85% ethanol was then applied for 24 h. The ethanol was removed and rinsed off with water after this period and replaced by lactic acid for 30–36 h before being washed with water. At 40x magnification, MCs and BSCs could be viewed. Measurements of leaf thickness, IVDs, numbers of MCs between veins and MC lengths were made for leaf 5–7 of three replicate plants per line. Figure 1 illustrates how these parameters could be taken from leaf section images.

Alkaline comet assays were performed using the Single Cell Gel Electrophoresis Assay Kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Briefly, cells were collected and suspended in ice cold PBS cells and then combined with molten LMAgarose (at 37 °C) at a ratio of 1:10 (v/v). The mixture was immediately pipetted onto CometSlide™ and incubated at 4 °C in the dark for 10 min. Then the slide was incubated with 4 °C lysis solution for 30–60 min and electrophoresis was performed. The slides were stained with DAPI and viewed under a fluorescence microscope BX51 equipped with a DP71 microscope digital camera (Olympus).

A ∼850-bp region including the t_MSE sequence (scaffold_4690:5995162..5996017 of the D. erecta reference genome rel. 1.3) was PCR amplified using forward (5′-TTCCGggcgcgccCCATGGAAGCCGAGCACCTGGTAGA-3′) and reverse primers (5′-TTGCCcctgcaggCTACAACGTRGGTCATGTNCAGGG-3′) with AscI and SbfI restriction sites given in small letters54 (link). PCR products were cloned into the S3aG vector55 (link), and inserted into the 51D site of D. melanogaster using ΦC31 integrase-mediated site-specific transgenesis (Best Gene Inc.)28 (link). Presence or absence of GFP expression of transgenic males and females were scored at eclosion under an Olympus SZX16 Stereo Microscope equipped with an Olympus DP71 microscope digital camera.