Imager a1 microscope

Ki67 stained sections (n = 6/group) were scanned using a Zeiss Imager A1microscope, fitted with a Prior Proscan II automatic stage (Zeiss, UK). For each scanned image 20 random fields were automatically selected by the programme and a 28 × 20 point grid was applied using Image Pro Plus 7.0 software. There were a total of 560 points (intersections of the grid) per field categorized as: (i) stained (positive) stromal cells, (ii) stained (positive) glandular cells, (iii) unstained (negative) stromal cells, (iv) unstained (negative) glandular cells and (v) all other points not overlaying any cells (e.g. empty slide space, lumen, connective tissue or blood). Points not overlaying a cell were excluded from analysis. The proportion of tissue in which each of the categorized cell type occupied was expressed as a percentage of total calculated area; cell proliferation index.

The wing scales were imaged on a Zeiss Axio Imager A1 microscope.

The results are shown as the means ± the SEM. The statistical analysis was performed with Student’s t-test or two-way ANOVA unless otherwise indicated. Significance was accepted at P < 0.05. For the quantitative analysis, all samples were evaluated at 200x magnification using an Imager A1 microscope (Zeiss) equipped with a digital camera (AxioCam MRc 5, Zeiss) and an image analyser (Image-Pro Plus^TM, Media Cybernetics, Silver Spring, MD, USA). The integrated optical density (IOD) was calculated for arbitrary areas in 10 fields with a standard area (0.04 mm²/field) per slide. Five sections were analysed per animal. The data obtained from the different experimental groups were analysed and scored blindly.

The following strains were incubated to mid-logarithmic phase with shaking (200 rpm) in YPD medium at either 30°C or 37°C, with or without the addition of 0.125 μM nocodazole: strains CNV108 (WT + GFP-Histone H4), CBN242 (WT + GFP- Tub1), CBN593(cdc420Δ + GFP-Histone H4), and CBN589 (cdc420Δ + GFP- Tub1). Nuclear size and division (noted by GFP-H4) and tubulin filaments (GFP-Tub1) were assessed using a Zeiss Axio Imager A1 microscope equipped with an Axio-Cam MRmdigital camera. Cells were imaged by DIC and with eGFP filter. Identical exposure times were used to image all cells. Fiji software [112 (link)] was used to process images.

For hyphal microscopy, conidia were inoculated onto precleaned glass coverslips overlaid with MMPDR liquid media. The strains were grown on coverslips at 37°C for various durations prior to observation under a microscope. The DNA was stained using 4,6-diamidino-2-phenylindole (DAPI) after fixing the cells with 4% paraformaldehyde (Polyscience, Warrington, PA) (42 (link), 43 (link)). Differential interference contrast (DIC) images and fluorescent images of the cells were collected using a Zeiss Axio imager A1 microscope (Zeiss, Jena, Germany).
For observation of conidiophore structure, the slide culture method for microscopic observation was performed as previously described (31 (link), 44 (link)), with a few modifications. Conidia were inoculated on the edge of a small square of agar medium placed on top of a coverslip, which was placed in a petri dish containing solidified agar to keep it moist. Another coverslip was placed on top of the agar square after inoculation. The coverslips with aerial hyphae and attached conidiophores were imaged using a SensiCam QE cooled digital camera system (Cooke Corporation, Germany) and analyzed with the MetaMorph/MetaFluor combination software package (Universal Imaging, West Chester, PA).